Immunogenic peptides

ABSTRACT

The invention provides relatively short immunogenic peptides, and biologically active variants thereof, associated with leukemia which elicit an immune response. Nucleic acids encoding the immunogenic peptides and antibodies specific for the peptides are also provided. The immunogenic peptides can be included in pharmaceutical compositions, such as cancer vaccines, and used for the treatment of cancer.

CLAIM FOR PRIORITY

This application claims priority to a provisional application Serial No.60/509,051 filed May 3, 2002, the entire contents of which are herebyincorporated by reference.

FIELD OF INVENTION

The present invention relates to immunogenic peptides. More particularlythe present invention relates to relatively short peptides associatedwith leukemia that elicit an immune response.

BACKGROUND OF THE INVENTION

Cancers, including leukemia, are the leading cause of death in humans.Roughly 32,000 new cases of, and 22,000 deaths caused by, leukemia occurin the U.S. each year. Most cases occur of leukemia occur in adults. Theexact cause of leukemia is not known, but links between certainactivities, such as exposure to carcinogens, and the incidence ofcertain types of carcinomas, lymphomas, e.g., leukemia and tumors, hasbeen shown by a number of researchers. However, such exposures do notexplain most cases of leukemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemiain adults, with an annual incidence of 2.7 per 100,000 adults in theU.S. (Murashige et al., 2002). Progress in therapy and supportive careover the past three decades has led to a gradual improvement in theoverall results, however, very little progress has been made regardinglong-term survival. Newly diagnosed AML patients achieve completeremission (CR) following chemotherapy/radiation treatment, with a medianduration of 15 months. A small percentage of patients with relapsed orrefractory AML can be induced with chemotherapy/radiation to achieve asecond CR and the duration of these responses are often only 4-8 months.Allogeneic stem cell transplantation (AlloSCT) has been conducted as asalvage therapy for those in whom conventional chemotherapy failed tocontrol their disease, but most of them are relapsed.

Many types of chemotherapeutic agents have been shown to be effectiveagainst leukemia, but not all types of leukemia cells respond to theseagents, and, unfortunately, many of these agents also destroy normalcells. Despite advances in the field of leukemia treatments, the leadingtherapies to date are radiation, chemotherapy and bone marrowtransplants. However, these therapies generally harm normal cells aswell as leukemic cells. Ideally cytotoxic agents that have specificityfor leukemia cells while only minimally affecting normal healthy cellswould be extremely desirable. Unfortunately, none have been found andinstead agents which target especially rapidly dividing cells (bothdiseased and normal) have been used.

Thus there continues to be a strong need for methods of diagnosing andviable treatment regimens for leukemia.

SUMMARY OF THE INVENTION

One aspect of the present invention provides isolated or recombinantleukemic antigens comprising a fragment of CD33 antigen or a variantthereof that is capable of stimulating a cytotoxic T-lymphocytereaction. The fragment or variant-thereof can be 8, 9, 10, 11 or 12amino acids in length up to 50 or 80 amino acids in length. In someembodiments, the first position of the fragment is Y and the otherpositions are variable. In these and other embodiments, the secondposition of the fragment is L, M or I. In these and other embodiments,the fourth position is E or K. Additionally, the eighth position of thefragment can be K. When the fragment is at least nine residues in lengththe ninth position of the fragment can be V, T, L or I. In some aspectsthe isolated leukemic antigen is immunologically recognized by MHCrestricted T-Lymphocytes that are HLA-A2.1 restricted. The identifiedleukemic antigen can have the amino acid sequence YLALCLCLI (SEQ ID NO:1), AIISGDSPV (SEQ ID NO: 2), YIISGDSPV (SEQ ID NO: 3), YIISGISPV (SEQID NO: 6), YLISGDSPV (SEQ ID NO: 7), ALLALCLCL (SEQ ID NO: 8), TIQLNVTYV(SEQ ID NO: 9), YIGSGDSPV (SEQ ID NO: 10), YIIIGDSPV (SEQ ID NO: 11),YIILGDSPV (SEQ ID NO: 12), YIISGDLPV (SEQ ID NO: 13), YIISGDSWV (SEQ IDNO: 14), YIISGDSPL (SEQ ID NO: 15), ALISGDSPV (SEQ ID NO: 16) orLLALCLCLI (SEQ ID NO:42) with or without one or more, such as one, two,two, three, four, five or more conservative or nonconservative aminoacid substitutions. The isolated leukemic antigen can also be combinedwith one or more co-immunostimulatory molecules.

The present invention also provides a method for stimulating an immuneeffector cell response achieved by contacting the isolated leukemicantigen with an immune effector cell which stimulates the immuneeffector cell to respond against the isolated leukemic antigen. In somemethods the immune effector cell is a naïve T-lymphocyte or a memoryT-lymphocyte. The method can be performed by contacting the isolatedleukemic antigen with an antigen presenting cell, in vivo or in vitro,such that the antigen presenting cell contacts the isolated leukemicantigen with the immune effector cell. Suitable antigen presenting cellsare dendritic cells or T2 cells.

The present invention also pertains to immune effector cells and antigenpresenting cells produced by these methods. Nucleic acids encoding thepresent isolated leukemic antigens also form part of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows IFN-γ production of cytotoxic T-lymphocytes (CTLs)stimulated by dendritic cells pulsed with the AIISGDSPV (SEQ ID NO: 2)peptide; and

FIG. 1B shows IFN-γ production of CTLs stimulated by T2 cells pulsedwith the AIISGDSPV (SEQ ID NO: 2) peptide.

FIG. 2 shows up-regulation of HLA-A2.1, CD80, CD86 expressions on AMLcells by cytokines treatment. Phenotype analysis was performed usinguntreated or cytokines (1,000 U/ml GM-CSF, 100 ng/ml IFN-γ, and 15 ng/mlTNF-a)-treated bone marrow blasts from HLA-A2.1+AML patients.Up-regulation of HLA-A2.1, CD80, and CD86, but not CD33 were observed onAML cells by the treatment of cytokines for two days. The values of meanfluorescence intensity are following (untreated vs. treated); 2198 vs.6005 for HLA-A2.1, 42 vs. 53 for CD33, 114 vs. 1208 for CD80, and 51 vs.267 for CD86 expression.

FIG. 3 shows the cytotoxic activity of peptide AIISGDSPV (SEQ ID NO:2)-specific CTLs against bone marrow blasts from two AML patients (A,B). CTLs were generated by stimulating T lymphocytes obtained from anHLA-A2.1+donor with autologus mature dendritic cells pulsed with thispeptide. Data shows the ⁵¹Cr release assay performed one week after thesecond stimulation at an Effector: Target cell ratio of 60:1. Resultsdemonstrate antigen-specific cytotoxicity against the untreated (whitebars) or GM-CSF/IFN-γ/TNF-α treated (black bars) bone marrow blasts fromboth AML patients.

FIG. 4 shows the cytotoxic activity of peptide AIISGDSPV (SEQ ID NO:2)-specific CTLs against the HLA-A2.1+ML-2 cells (AML cell line). CTLswere generated by stimulating T lymphocytes obtained from six differentHLA-A2.1+ donors with either autologus mature DCs (A, B, C, D) or T2 (E,F) cells pulsed with this peptide. Data shows the ⁵¹Cr release assayperformed one week after the last stimulation at an Effector:Target cellratio of 60:1 (white bars—unstimulated, black bars—CTLs stimulatedtwice, hatched bars—CTLs stimulated three times). Results demonstratehigh levels of antigen-specific cytotoxicities against cytokine(GM-CSF/IFN-γ/TNF-α) treated ML-2 cells by CD33-specific CTLs.Increasing number of stimulations of the CTLs enhanced the level ofcytotoxicity against the target cells.

FIG. 5 shows the cytotoxic activity of CD33 peptide-specific CTLsagainst normal peripheral blood mononuclear cells (PBMCs) or monocytes.CD33 peptide-specific CTLs were generated by stimulating HLA-A2.1+ Tlymphocytes with autologus mDCs pulsed with CD33 peptide. Assays wereperformed one week after the second stimulation to test the cytolyticactivities of the CTLs against normal allogeneic HLA-A2.1+ PBMCs ormonocytes (donor 1, 2). No significant cytotoxicities were observed bythe CTLs at different ratios of Target: Effector.

FIG. 6 shows the cytotoxicity of AIISGDSPV (SEQ ID NO: 2)peptide-specific CTLs evaluated in a Cold Target Inhibition assay. CTLswere incubated for 1 hour with “cold” unlabeled ML-2 cells beforeincubation with ⁵¹Cr labeled AML bone marrow blasts (white bars) or ML-2cells (black bars). Results demonstrate the inhibition of cytotoxicityby the CD33+ HLA A2.1+“cold inhibitor” ML-2 cells against theCD33+HLA-A2.1+51^(Cr)-labeled target cells, and thus the cytotoxicity ofthe CTLs was AML cell-specific.

FIG. 7 shows the identification of tetramer-reactive CD8+ T lymphocytesbefore and after in vitro AIISGDSPV (SEQ ID NO: 2) peptide-specificstimulation. T-lymphocytes from HLA-A2.1+ normal donors after A) CD33peptide-specific stimulation (3×), B) unstimulated HLA-A2.1+, or C)unstimulated HLA-A2.1− T lymphocytes were stained with anti-CD8-FITC andPE-conjugated CD33-AIISGDSPV-HLA-A2.1 tetrameric complexes. Thepercentage of tetramer+CD8+ T cells are shown in the upper rightquadrant of the dot plots.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention encompasses fragments of the CD33 antigen,variants, isoforms and other mammalian homologs thereof which areimmunologically recognized by T lymphocytes of the immune system. CD33antigen is generally expressed by myeloblasts, promyelocytes,myeolcytes, mast cells, throughout monocyte differentiation and is alsohighly expressed on a large percentage of leukemic cells. The presentinvention further encompasses the antigen cancer epitope(s) which arecontained in the tumor antigen. The antigenic cancer epitopespecifically causes a cellular mediated immune response by interactionwith T cells of the immune system. This interaction between theantigenic cancer epitope and the T cells causes the T cells to respondagainst, and prevent, eliminate or reduce the cancer in a mammal,including humans. The peptides, nucleic acid molecules which code forsuch peptides, binding agents such as antibodies, antigen presentingcells (APCs) and/or immune system cells (e.g., T cells), antibodiesagainst such peptides and nucleic acids, are useful, inter alia, indiagnostic and therapeutic contexts. Thus, the present inventionprovides a cancer vaccine.

CD33 is a member of the sialic acid-binding receptor family and isexpressed on AML blast cells in greater than 90% of AML cases. Thisantigen is also expressed on normal myeloid precursor cells resulting inneutropenia and thrombocytopenia associated with anti-CD33 therapy.However, CD33 is not expressed on pluripotent stem cells, which allowshematologic recovery after the treatment period. The CD33 antigen isdiscussed in Simmons et al., J. Immunol. 141 (8), 2797-2800 (1988).Proteins similar or homologous to CD33 are discussed in Tchilian et al.Blood 83 (11), 3188-3198 (1994); Takei et al. Cytogenet. Cell Genet. 78(3-4), 295-300 (1997). The sequence for the CD33 antigen) as reported inSimmons et al. and disclosed in the SwissProt annotated protein recordP20138 is as follows:

-   -   1 mplllllpll wagalamdpn fwlqvqesvt vqeglcvlvp ctffhpipyy        dknspvhgyw    -   61 fregaiisgd spvatnkldq evqeetqgrf rllgdpsrnn cslsivdarr        rdngsyfform    -   121 ergstkysyk spqlsvhvtd lthrpkilip gtlepghskn ltcsvswace        qgtppifswl    -   181 saaptslgpr tthssvliit prpqdhgtnl tcqvkfagag vttertiqln        vtyvpqnptt    -   241 gifpgdgsgk qetragvvhg aiggagvtal lalcicliff ivkthrrkaa        rtavgrndth    -   301 pttgsaspkh qkksklhgpt etsscsgaap tvemdeelhy aslnfhgmnp        skdtsteyse    -   361 vrtq        (SEQ ID NO: 4).

References discussing the CD33 protein, sequence, nucleic acids encodingand antibodies against include: Andrews et al. Blood 62:124-132 (1983);Andrews et al., J Exp Med 169:1721-1731 (1989); Caron et al. Blood83:1760-1768 (1994); Freeman et al., Blood 85:2005-2012 (1995); Griffinet al., Leuk Res 8:521-534 (1984); Peiper et al., Leukocyte Typing V.Oxford University Press p837-840 (1995); Peiper et al., Blood 72:314-321(1988); Robertson et al., Blood 79:2229-2236 (1992); Sobol et al., NEngl J Med 316:1111-1117 (1987); and Thomas et al., J Immunol153:4016-4028 (1994).

The domains or regions of CD33 are as follows:

-   -   (1) Signal peptide (aa 1-17);    -   (2) Extracellular region of 241 residues that includes and IgV        domain (aa 18-121) and an IgC2 type domain (aa 156-219);    -   (3) Transmembrane spanning domain (aa 260-282); and    -   (4) Cytoplasmic tail (aa 283-364)

Proteins

The compounds of this invention generally comprise a polypeptide,sometimes in isolated form, that stimulates a Th1 or CTL (cytotoxicT-lymphocyte) immune response in peripheral blood mononuclear cells(PBMCs). In particular, polypeptides 8-12 amino acids in lengthcomprising a stimulatory portion of the CD33 antigen are disclosed, suchas a cancer rejection antigen. The peptides of the present invention canalso be from 8 to 80 amino acids in length, such as 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20, 25, 30, 32, 35, 40, 45, 50, 55, 60,65, 70, 75 or 80 residues, or consecutive amino acids of CD33, inlength. Preferably, when the peptide is longer in length, then thesequence of the peptide will substantially correspond with the sequenceof CD33, for example within about 1, 2, 5, 10 or 20 percent homology oridentity. A cancer rejection antigen is an example of a unique fragmentof a cancer specific polypeptide which retains the functional capabilityof HLA binding and interaction with cytotoxic T lymphocytes. Tumorrejection antigens presented by HLA molecules typically are 9 aminoacids in length, although peptides of 8, 9, 10, 11 and 12 and more aminoacids, up to about 80, can retain the capability to interact with HLAand cytotoxic T lymphocyte to an extent effective to provoke a cytotoxicT lymphocyte response (see, e.g., Van den Eynde & Brichard, Curr. Opin.Immunol. 7:674-681, 1995; Coulie et al., Stem Cells 13:393-403, 1995)and discussed in U.S. Pat. No. 6,271,019. Polypeptides encompass aminoacid chains of any length, including full length proteins and portionsthereof, wherein amino acid residues are linked by covalent peptidebonds. Although CD33 fragments are described herein for exemplarypurposes, portions thereof, variants of the polypeptide (or portionsthereof) and homologous proteins in other mammals can also be used. Inone preferred embodiment, the polypeptides are substantially free ofcontaminating endogenous materials. In some embodiments, the peptidesare derived from the signalling domain, extracellular domain,transmembrane spanning domain or cytoplasmic tail of CD33.

The term “isolated” means separated from constituents, cellular andotherwise, in which the polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, are normally associated with in nature.As is apparent to those of skill in the art, a non-naturally occurringpolynucleotide, peptide, polypeptide, protein, antibody, or fragmentsthereof, does not require “isolation” to distinguish it from itsnaturally occurring counterpart. In addition, a “concentrated,”“separated” or “diluted” polynucleotide, peptide, polypeptide, protein,antibody, or fragment thereof, is distinguishable from its naturallyoccurring counterpart in that the concentration or number of moleculesper volume is greater than “concentrated” or less than “separated” thanthat of its naturally occurring counterpart. A polynucleotide, peptide,polypeptide, protein, antibody, or fragments thereof, which differs fromthe naturally occurring counterpart in its primary sequence or forexample, by its glycosylation pattern, need not be present in itsisolated form since it is distinguishable from its naturally occurringcounterpart by its primary sequence, or alternatively, by anothercharacteristic such as glycosylation pattern. Although not explicitlystated for each of the inventions disclosed herein, it is to beunderstood that all of the above embodiments for each of thecompositions disclosed below and under the appropriate conditions, areprovided by this invention. Thus, a non-naturally occurringpolynucleotide is provided as a separate embodiment from the isolatednaturally occurring polynucleotide. A protein produced in a bacterialcell is provided as a separate embodiment from the naturally occurringprotein isolated from a eukaryotic cell in which it is produced innature.

Several peptide fragments of the CD33 antigen and have been discoveredwhich have the ability to stimulate a T-lymphocyte mediated cellularimmune response. Peptides of the present invention can include theseamino acid sequences in any configuration or location in the peptide. Insome embodiments, specific positions in the peptides, referred tohereinafter as anchor positions, have defined amino acids. Examples ofsuch anchor positions can include Y as the first amino acid of thepeptide sequence with the other positions being variable. Other anchorpositions include the second position of the fragment which can be L, Mor I, the fourth position which can be E, K or S. Additionally, theeighth position of the fragment can be an anchor position and can be K.When the fragment is at least nine residues in length, the first, secondand ninth positions of the fragment can be anchor positions in which thefirst position can be Y, the second can be L or M, and/or the ninthposition can be V or L. The peptides disclosed herein can have one ormore of any of these anchor positions with the recited amino acids.Accordingly, the present peptides cover all possible combinations andsubcombinations of these anchor sequences. Preferably, fragments of theCD33 protein having one or more of these anchor positions, such as thoseshown below, are used in the present methods and compositions. Suchpeptides can have these following sequences, where a 1 indicates thepresence of an anchor amino acid at the first position as recited above,i.e. a Y, a 2 indicates the presence of an anchor amino acid at thesecond position as recited above, i.e. a L, M or I, a 4 indicates thepresence of an anchor amino acid at the fourth position as recitedabove, i.e. a E, K or S, an 8 indicates the presence of an anchor aminoacid at the eighth position as recited above, i.e. a K, and a 9indicates the presence of an anchor amino acid at the ninth position asrecited above, i.e. a V, T, L or I, X indicates the presence of anyamino acid residue and Xa indicates an optional amino residue.

1XXXXXXX (SEQ ID NO: 17) X2XXXXXX (SEQ ID NO: 18) XXX4XXXX (SEQ ID NO:19) XXXXXXX8 (SEQ ID NO: 20) XXXXXXXX9 (SEQ ID NO: 21) 12XXXXXX (SEQ IDNO: 22) 1XX4XXXX (SEQ ID NO: 23) 1XXXXXX8a (SEQ ID NO: 24) 1XXXXXXX9(SEQ ID NO: 25) 12X4XXXX (SEQ ID NO: 26) 12XXXXX8 (SEQ ID NO: 27)12XXXXXX9 (SEQ ID NO: 28) 12X4XXX8 (SEQ ID NO: 29) 12X4XXXX9 (SEQ ID NO:30) 12X4XXX89 (SEQ ID NO: 31) X2X4XXXX (SEQ ID NO: 32) X2XXXXX8 (SEQ IDNO: 33) X2XXXXXX9 (SEQ ID NO: 34) X2X4XXX8 (SEQ ID NO: 35) X2X4XXXX9(SEQ ID NO: 36) X2X4XXX89 (SEQ ID NO: 37) XXX4XXX8 (SEQ ID NO: 38)XXX4XXXX9 (SEQ ID NO: 39) XXX4XXX89 (SEQ ID NO: 40) XXXXXXX89 (SEQ IDNO: 41)

The present peptides should be capable of stimulating a cytotoxicT-lymphocyte reaction. These anchor positions also provide a guide tothe skilled artisan for modifying fragments of CD33 to increase theimmunogenicity of the peptide. As will be understood by one skilled inthe art, generally, the first, second, and ninth amino acids areconsidered to be important for binding to MHC molecules and the third,fourth, fifth, sixth and seventh amino acids are considered as importantfor recognition by T-cell receptors. However, the interactions withneighboring amino acids are also important to MHC bindings andrecognition by T-cell receptors. As will be understood by the skilledartisan, the anchor positions denoted above are only numbered withrespect to one another but can be placed in any relative orientation orappropriate place within a larger immunogenic peptide. For example, SEQID NO:30 can be part of 25 amino acid peptide and anchor position 1 canstart at position 5, 7, 8, etc. within the peptide. Preferably, peptideshaving these anchor positions have sequences at the non-anchor positionsthat correspond to the CD33 sequence. Thus target fragments of CD33 andmammalian homologs thereof can be identified by aligning the desiredanchor position or positions with the CD33 or homologous proteinsequence and selecting the desired portion of the CD33 sequence orhomolog.

Specific examples of peptides include peptides as discussed hereininclude the amino acid sequence YLALCLCLI (SEQ ID NO: 1), AIISGDSPV (SEQID NO: 2), YIISGDSPV (SEQ ID NO: 3), YIISGISPV (SEQ ID NO: 6), YLISGDSPV(SEQ ID NO: 7), ALLALCLCL (SEQ ID NO: 8), TIQLNVTYV (SEQ ID NO: 9),YIGSGDSPV (SEQ ID NO: 10), YIIIGDSPV (SEQ ID NO: 11), YIILGDSPV (SEQ IDNO: 12), YIISGDLPV (SEQ ID NO: 13), YIISGDSWV (SEQ ID NO: 14), YIISGDSPL(SEQ ID NO: 15), ALISGDSPV (SEQ ID NO: 16) or LLALCLCLI (SEQ ID NO:42)with or without one or more conservative or nonconservative amino acidsubstitutions. Combinations of these peptides are also suitable for usein the present compounds and methods described herein.

Biologically functionally equivalent variants of the present CD33polypeptide fragments, i.e., variants of polypeptides which retain thefunction of the natural polypeptide fragment, can be prepared accordingto methods for altering polypeptide sequence known to one of ordinaryskill in the art such as are found in references which compile suchmethods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, etal., eds., Second Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. The skilledartisan will also realize that conservative amino acid substitutions canbe made in the present polypeptides to provide such functionally activehomologs of the forgoing polypeptides, i.e., the homologs retain thefunctional capabilities of the polypeptides. As used herein, a“conservative amino acid substitution” refers to an amino acidsubstitution which does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Conservative substitutions of amino acids generally are understoodto include substitutions made amongst amino acids within the followinggroups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T;(f) Q, N; and (g) E, D. The present invention also encompassespolypeptides with one or more nonconservative amino acid substitutionsthat retain similar functionality compared to the non-modified peptide.Generally a “nonconservative amino acid substitution” is understood tobe an amino acid substituted by an alternative amino acid of differingcharge density, hydrophilicity/hydrophobicity, size, and/orconfiguration (e.g., Val for Phe). The means of making suchmodifications are well known in the art and can be readily accomplishedby means of commercially available kits and vectors (e.g., New EnglandBiolabs, Inc., Beverly, Mass.; Clontech, Palo Alto, Calif.). Moreover,the means of assessing such substitutions (e.g., in terms of effect onability to bind and enter cells) are known in the art and described forexample in U.S. Pat. No. 6,329,190.

The polypeptides of the present invention also include variants of theCD33 fragments that retain the ability to stimulate a Th1 or CTL immuneresponse in PBMCs. Such variants include various structural forms of theprimary protein, including related and homologous proteins which can befound in non-human species. Due to the presence of ionizable amino andcarboxyl groups, for example, a polypeptide fragment can be in the formof an acidic or basic salt, or can be in neutral form. Individual aminoacid residues can also be modified by oxidation or reduction.

Variants within the scope of this invention also include polypeptides inwhich the primary amino acid structure of the polypeptide fragment ismodified by forming covalent or aggregative conjugates with otherpolypeptides or chemical moieties such as glycosyl groups, lipids,phosphate, acetyl groups and the like. Covalent derivatives can beprepared, for example, by linking particular functional groups to aminoacid side chains or at the N- or C-termini. Alternatively, forderivatives in which a polypeptide is joined to a CD33 fragment, afusion protein can be prepared using recombinant DNA techniques, asdescribed below. As will be understood by the skilled artisan, fusionpeptides containing the 8-12 amino acid CD33 fragment of the presentinvention are not limited to 8-12 amino acids in total size, but insteadthat the immunogenic fragment of the CD33 antigen is 8-12 residues insize. In one such embodiment, the CD33 polypeptide polypeptide can beconjugated to a signal (or leader) polypeptide sequence at theN-terminal region of the protein which co-translationally orpost-translationally directs transfer of the protein from its site ofsynthesis to its site of function inside or outside of the cell membraneor wall (e.g., the yeast α-factor leader).

The present peptides can also be derived from non-human mammalianpeptides that are homologous to CD33. Examples of murine CD33 homologsare disclosed in Tchilian et al. Blood Jun. 1, 1994;83(11):3188-98.

Also provided by this application are the polypeptides and proteinsdescribed herein conjugated to a detectable agent for use in thediagnostic methods. For example, detectably labeled proteins andpolypeptides can be bound to a column and used for the detection andpurification of antibodies. They also are useful as immunogens for theproduction of antibodies as described below. The proteins and fragmentsof this invention are useful in an in vitro assay system to screen foragents or drugs, which modulate cellular processes.

Such detectable agents include protein fusions with other proteins andwhich can facilitate purification or identification of the polypeptides(e.g., poly-His). For example, the peptide described by Hopp et al.,Bio/Technology 6:1204 (1988) is a highly antigenic peptide that can beused to facilitate identification. Such a peptide provides an epitopereversibly bound by a specific monoclonal antibody, enabling rapid assayand facile purification of expressed recombinant protein. The sequenceof Hopp et al. is also specifically cleaved by bovine mucosalenterokinase, allowing removal of the peptide from the purified protein.Fusion proteins capped with such peptides can also be resistant tointracellular degradation in E. coli.

Protein fusions encompassed by this invention further include, forexample, the polypeptides linked to an immunoglobulin Fe region. If thefusion proteins are made with both heavy and light chains of anantibody, it is possible to form a protein oligomer with as many as fourCD33 protein fragment regions. Also within the scope of the presentinvention are polypeptides linked to a leucine zipper domain. Leucinezipper domains are described, for example, in published PCT ApplicationWO 94/10308. The present polypeptides comprising leucine zippers may,for example, be oligomeric, dimeric or trimeric. All of the aboveprotein fusions can be prepared by chemical linkage or as fusionproteins, as described in U.S. Pat. No. 6,013,268. Preferred proteinfusions include polypeptides that comprise sequences useful forstimulating immunity to infectious pathogens (e.g., antigens). Suchsequences can be derived, for example, from viruses, tumor cells,parasites or bacteria.

The proteins and polypeptides of this invention can be obtained bychemical synthesis using a commercially available automated peptidesynthesizer such as those manufactured by Perkin Elmer/AppliedBiosystems, Inc., Model 430A or 431A, Foster City, Calif., USA. Thesynthesized protein or polypeptide can be precipitated and furtherpurified, for example by high performance liquid chromatography (HPLC).Accordingly, this invention also provides a process for chemicallysynthesizing the proteins of this invention by providing the sequence ofthe protein and reagents, such as amino acids and enzymes and linkingtogether the amino acids in the proper orientation and linear sequence.

Alternatively, the proteins and polypeptides can be obtained bywell-known recombinant methods as described above using the host celland vector systems described above.

Nonpeptide analogs of peptides, e.g., those which provide a stabilizedstructure or lessened biodegradation, are also contemplated. Peptidemimetic analogs can be prepared based on a selected binding peptide byreplacement of one or more residues by nonpeptide moieties. Preferably,the nonpeptide moieties permit the peptide to retain its naturalconformation, or stabilize a preferred, e.g., bioactive, confirmation.Such peptides can be tested in molecular or cell-based binding assays toassess the effect of the substitution(s) on conformation and/oractivity. One example of methods for preparation of nonpeptide mimeticanalogs from peptides is described in Nachman et al., Regul. Pept.57:359-370 (1995) and disclosed in U.S. Pat. No. 6,291,430.

The proteins of this invention also can be combined with various liquidphase carriers, such as sterile or aqueous solutions, pharmaceuticallyacceptable carriers, suspensions and emulsions. Examples of non-aqueoussolvents include propyl ethylene glycol, polyethylene glycol andvegetable oils. When used to prepare antibodies, the carriers also caninclude an adjuvant that is useful to non-specifically augment aspecific immune response. A skilled artisan can easily determine whetheran adjuvant is required and select one. However, for the purpose ofillustration only, suitable adjuvants include, but are not limited toFreund's Complete and Incomplete, mineral salts, polynucleotides, GM-CSFand Keyhole Limpet Hemocyanin (KLH). Adjuvant can be used as is known inthe art, for example as a carrier or vehicle.

Nucleic Acids

Polynucleotides of the subject invention generally comprise a DNA or RNAsequence that encodes all or a portion of the above CD33 polypeptidefragments, or that is complementary to such a sequence. Nucleic acidsencoding proteins related or homologous to CD33 are disclosed inTchilian et al. Blood 83 (11), 3188-3198 (1994); and Takei et al.Cytogenet. Cell Genet. 78 (3-4), 295-300 (1997). Preferably, the CD33polypeptide fragment is a leukemia associated nucleic acid orpolypeptide is a nucleic acid or polypeptide expressed preferentially inleukemias and solid forms of leukemia cell malignancies, such aslymphomas. Various methods for determining the expression of a nucleicacid and/or a polypeptide in normal and leukemia cells are known tothose of skill in the art.

The reported nucleic acid sequence encoding the CD33 protein describedabove, as reported in Simmons et al., J. Immunol. 141 (8), 2797-2800(1988), is (SEQ ID NO: 5):

-   -   1 gcttcctcag acatgccgct gctgctactg ctgcccctgc tgtgggcagg        ggccctggct    -   61 atggatccaa atttctggct gcaagtgcag gagtcagtga cggtacagga        gggtttgtgc    -   121 gtcctcgtgc cctgcacttt cttccatccc ataccctact acgacaagaa        ctccccagtt    -   181 catggttact ggttccggga aggagccatt atatccgggg actctccagt        ggccacaaac    -   241 aagctagatc aagaagtaca ggaggagact cagggcagat tccgcctcct        tggggatccc    -   301 agtaggaaca actgctccct gagcatcgta gacgccagga ggagggataa        tggttcatac    -   361 ttctttcgga tggagagagg aagtaccaaa tacagttaca aatctcccca        gctctctgtg    -   421 catgtgacag acttgaccca caggcccaaa atcctcatcc ctggcactct        agaacccggc    -   481 cactccaaaa accttacctg ctctgtgtcc tgggcctgtg agcagggaac        acccccgatc    -   541 ttctcctggt tgtcagctgc ccccacctcc ctgggcccca ggactactca        ctcctcggtg    -   601 ctcataatca ccccacggcc ccaggaccac ggcaccaacc tgacctgtca        ggtgaagttc    -   661 gctggagctg gtgtgactac ggagagaacc atccagctca acgtcaccta        tgttccacag    -   721 aacccaacaa ctggtatctt tccaggagat ggctcaggga aacaagagac        cagagcagga    -   781 ctggttcatg gggccattgg aggagctggt gttacagccc tgctcgctct        ttgtctctgc    -   841 ctcatcttct tcatagtgaa gacccacagg aggaaagcag ccaggacagc        agtgggcagc    -   901 aatgacaccc accctaccac agggtcagcc tccccgaaac accagaagaa        ctccaagtta    -   961 catggcccca ctgaaacctc aagctgttca ggtgccgccc ctactgtgga        gatggatgag    -   1021 gagctgcatt atgcttccct caactttcat gggatgaatc cttccaagga        cacctccacc    -   1081 gaatactcag aggtcaggac ccagtgagga accctcaaga gcatcaggct        cagctagaag    -   1141 atccacatcc tctacaggtc ggggaccaaa ggctgattct tggagattta        actccccaca    -   1201 ggcaatgggt ttatagacat tatgtgagtt tcctgctata ttaacatcat        cttgagactt    -   1261 tgcaagcaga gagtcgtgga atcaaatctg tgctctttca tttgctaagt        gtatgatgtc    -   1321 acacaagctc cttaaccttc catgtctcca ttttcttctc tgtgaagtag        gtataagaag    -   1381 tcctatctca tagggatgct gtgagcatta aataaaggta cacatggaaa        acaccag

The nucleic acids contemplate the degeneracy of the genetic code inwhich nucleic acids can be coded by alternative codons to those presentin the native materials. For example, serine residues are encoded by thecodons TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons isequivalent for the purposes of encoding a serine residue. Thus, it willbe apparent to one of ordinary skill in the art that any of theserine-encoding nucleotide triplets can be employed to direct theprotein synthesis apparatus, in vitro or in vivo, to incorporate aserine residue. Similarly, nucleotide sequence triplets which encodeother amino acid residues include, but are not limited to: CCA, CCC, CCGand CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (argininecodons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT(asparagine codons); and ATA, ATC and ATT (isoleucine codons). Otheramino acid residues can be encoded similarly by multiple nucleotidesequences. Thus, the invention embraces degenerate nucleic acids thatdiffer from the biologically isolated nucleic acids in codon sequencedue to the degeneracy of the genetic code.

Further contemplated are antisense oligonucleotides that selectivelybind to a leukemia associated gene nucleic acid molecule. Additionally,nucleic acid mimetics, such as peptide nucleic acids are contemplated inthe definition of nucleic acids.

The polynucleotides and peptides can be used for comparison to known andunknowns sequences using a computer-based method to match a samplesequence with known sequences. Thus, this invention also provides thepolynucleotides or peptides in a computer database or in computerreadable form, including applications utilizing the internet.

A linear search through such a database can be used. Alternatively, thepolynucleotide sequence can be converted into a unique numericrepresentation. The comparison aspects can be implemented in hardware orsoftware, or a combination of both. Preferably, these aspects of theinvention are implemented in computer programs executing on aprogrammable computer comprising a processor, a data storage system(including volatile and non-volatile memory and/or storage elements), atleast one input device, and at least one output device. Data inputthrough one or more input devices for temporary or permanent storage inthe data storage system includes sequences, and can include previouslygenerated polynucleotides and codes for known and/or unknown sequences.Program code is applied to the input data to perform the functionsdescribed above and generate output information. The output informationis applied to one or more output devices, in known fashion.

Each such computer program is preferably stored on a storage media ordevice (e.g., ROM or magnetic diskette) readable by a general or specialpurpose programmable computer, for configuring and operating thecomputer when the storage media or device is read by the computer toperform the procedures described herein. The inventive system can alsobe considered to be implemented as a computer-readable storage medium,configured with a computer program, where the storage medium soconfigured causes a computer to operate in a specific and predefinedmanner to perform the functions described herein.

The polynucleotides of the present invention also can serve as primersfor the detection of genes or gene transcripts that are expressed inAPC, for example, to confirm transduction of the polynucleotides intohost cells. In this context, amplification means any method employing aprimer-dependent polymerase capable of replicating a target sequencewith reasonable fidelity. Amplification can be carried out by natural orrecombinant DNA-polymerases such as T7 DNA polymerase, Klenow fragmentof E. coli DNA polymerase, and reverse transcriptase.

The invention further provides the isolated polynucleotide operativelylinked to a promoter of RNA transcription, as well as other regulatorysequences for replication and/or transient or stable expression of theDNA or RNA. As used herein, the term “operatively linked” meanspositioned in such a manner that the promoter will direct transcriptionof RNA off the DNA molecule. Examples of such promoters are SP6, T4 andT7. In certain embodiments, cell-specific promoters are used forcell-specific expression of the inserted polynucleotide. Vectors whichcontain a promoter or a promoter/enhancer, with termination codons andselectable marker sequences, as well as a cloning site into which aninserted piece of DNA can be operatively linked to that promoter arewell known in the art and commercially available. For generalmethodology and cloning strategies, see GENE EXPRESSION TECHNOLOGY(Goeddel ed., Academic Press, Inc. (1991)) and references cited thereinand VECTORS: ESSENTIAL DATA SERIES (Gacesa and Ramji, eds., John Wiley &Sons, N.Y. (1994)), which contains maps, functional properties,commercial suppliers and a reference to GenEMBL accession numbers forvarious suitable vectors. Preferable, these vectors are capable oftranscribing RNA in vitro or in vivo.

Expression vectors containing these nucleic acids are useful to obtainhost vector systems to produce proteins and polypeptides. It is impliedthat these expression vectors must be replicable in the host organismseither as episomes or as an integral part of the chromosomal DNA.Suitable expression vectors include plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, cosmids, etc.Adenoviral vectors are particularly useful for introducing genes intotissues in vivo because of their high levels of expression and efficienttransformation of cells both in vitro and in vivo. When a nucleic acidis inserted into a suitable host cell, e.g., a prokaryotic or aeukaryotic cell and the host cell replicates, the protein can berecombinantly produced. Suitable host cells will depend on the vectorand can include mammalian cells, animal cells, human cells, simiancells, insect cells, yeast cells, and bacterial cells constructed usingwell known methods. See Sambrook, et al. (1989) Supra. In addition tothe use of viral vector for insertion of exogenous nucleic acid intocells, the nucleic acid can be inserted into the host cell by methodswell known in the art such as transformation for bacterial cells;transfection using calcium phosphate precipitation for mammalian cells;or DEAE-dextran; electroporation; or microinjection. See Sambrook et al.(1989) Supra for this methodology. Thus, this invention also provides ahost cell, e.g. a mammalian cell, an animal cell (rat or mouse), a humancell, or a prokaryotic cell such as a bacterial cell, containing apolynucleotide encoding a protein or polypeptide or antibody.

When the vectors are used for gene therapy in vivo or ex vivo, apharmaceutically acceptable vector is preferred, such as areplication-incompetent retroviral or adenoviral vector.Pharmaceutically acceptable vectors containing the nucleic acids of thisinvention can be further modified for transient or stable expression ofthe inserted polynucleotide. As used herein, the term “pharmaceuticallyacceptable vector” includes, but is not limited to, a vector or deliveryvehicle having the ability to selectively target and introduce thenucleic acid into dividing cells. An example of such a vector is a“replication-incompetent” vector defined by its inability to produceviral proteins, precluding spread of the vector in the infected hostcell. An example of a replication-incompetent retroviral vector is LNL6(Miller, A. D. et al. (1989) BioTechniques 7:980-990). The methodologyof using replication-incompetent retroviruses for retroviral-mediatedgene transfer of gene markers is well established (Correll, et al.(1989) PNAS USA 86:8912; Bordignon (1989) PNAS USA 86:8912-52; Culver,K. (1991) PNAS USA 88:3155; and Rill, D. R. (1991) Blood79(10):2694-700.

These host cells containing the polynucleotides of this invention areuseful for the recombinant replication of the polynucleotides and forthe recombinant production of peptides. Alternatively, the cells can beused to induce an immune response in a subject in the methods describedherein. When the host cells are antigen presenting cells, they can beused to expand a population of immune effector cells such as tumorinfiltrating lymphocytes which in turn are useful in adoptiveimmunotherapies.

Protein Binding Agents (Antibodies)

The invention also involves agents which bind to leukemia associatedpolypeptides disclosed herein. Such binding partners can be used inscreening assays to detect the presence or absence of the presentpolypeptides and in purification protocols to isolate thesepolypeptides. Likewise, such binding partners can be used to selectivelytarget drugs, toxins or other molecules to leukemia cells which presentthe associated polypeptides. In this manner, cells present in solid ornon-solid tumors which express the CD33 fragments can be treated withcytotoxic compounds.

The invention, therefore, involves antibodies or fragments of antibodieshaving the ability to selectively bind to the disclosed polypeptides.Antibodies against the CD33 antigen are discussed in Co et al., Chimericand Humanized Antibodies with Specificity for the CD33 Antigen, J.Immunol. 148, 1149-1154 (1992). Antibodies include polyclonal andmonoclonal antibodies, prepared according to conventional methodology.The antibodies can include, but are not limited to mouse, rat, andrabbit or human antibodies. The antibodies are useful to identify andpurify polypeptides and APCs expressing the polypeptides.

The antibodies of the present invention are prepared by any of a varietyof methods, including administering protein, fragments of protein, cellsexpressing the protein or fragments thereof and the like to an animal toinduce polyclonal antibodies. Laboratory methods for producingpolyclonal antibodies and monoclonal antibodies, as well as deducingtheir corresponding nucleic acid sequences, are known in the art, seeHarlow and Lane (1988) Supra and Sambrook, et al. (1989) Supra. Themonoclonal antibodies of this invention can be biologically produced byintroducing protein or a fragment thereof into an animal, e.g., a mouseor a rabbit. The antibody producing cells in the animal are isolated andfused with myeloma cells or heteromyeloma cells to produce hybrid cellsor hybridomas. Accordingly, the hybridoma cells producing the monoclonalantibodies of this invention also are provided.

The antibodies of this invention can be linked to a detectable agent orlabel. There are many different labels and methods of labeling known tothose of ordinary skill in the art. The coupling of antibodies to lowmolecular weight haptens can increase the sensitivity of the assay. Thehaptens can then be specifically detected by means of a second reaction.For example, it is common to use haptens such as biotin, which reactsavidin, or dinitropherryl, pyridoxal, and fluorescein, which can reactwith specific anti-hapten antibodies. See Harlow and Lane (1988) Supra.Antibodies also can be coupled to specific labeling agents for imagingor to antitumor agents, including, but not limited to, methotrexate,radioiodinated compounds, toxins such as ricin, other cytostatic orcytolytic drugs, and so forth. Antibodies prepared according to theinvention also preferably are specific for the CD33 complexes describedherein. Variations of antibodies encompasses by the present inventioncan be found in U.S. Pat. No. 6,303,756.

The monoclonal antibodies of the invention also can be bound to manydifferent carriers. Thus, this invention also provides compositionscontaining the antibodies and another substance, active or inert.Examples of well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses and magnetite. The natureof the carrier can be either soluble or insoluble for purposes of theinvention. Those skilled in the art will know of other suitable carriersfor binding monoclonal antibodies, or will be able to ascertain such,using routine experimentation.

Thus, using the protein or fragment thereof, and well known methods, oneof skill in the art can produce and screen the hybridoma cells andantibodies of this invention for antibodies having the ability to bindthe proteins or polypeptides. As detailed herein, such antibodies canalso be used to identify tissues expressing protein or to purifyprotein.

If a monoclonal antibody being tested binds with the protein orpolypeptide, then the antibody being tested and the antibodies providedby the hybridomas of this invention are equivalent. It also is possibleto determine without undue experimentation, whether an antibody has thesame specificity as the monoclonal antibody of this invention bydetermining whether the antibody being tested prevents a monoclonalantibody of this invention from binding the protein or polypeptide withwhich the monoclonal antibody is normally reactive. If the antibodybeing tested competes with the monoclonal antibody of the invention asshown by a decrease in binding by the monoclonal antibody of thisinvention, then it is likely that the two antibodies bind to the same ora closely related epitope. Alternatively, one can pre-incubate themonoclonal antibody of this invention with a protein with which it isnormally reactive, and determine if the monoclonal antibody being testedis inhibited in its ability to bind the antigen. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or a closely related, epitopic specificity as the monoclonalantibody of this invention.

The term “antibody” also is intended to include antibodies of allisotypes. Particular isotypes of a monoclonal antibody can be preparedeither directly by selecting from the initial fusion, or preparedsecondarily, from a parental hybridoma secreting a monoclonal antibodyof different isotype by using the sib selection technique to isolateclass switch variants using the procedure described in Steplewski, etal. (1985) Proc. Natl. Acad. Sci. 82:8653 or Spira, et al. (1984) J.Immunol. Methods 74:307.

This invention also provides biological active fragments of thepolyclonal and monoclonal antibodies described above. These “antibodyfragments” retain some ability to selectively bind with its antigen orimmunogen. Such antibody fragments can include, but are not limited to:(1) Fab, (2) Fab′, (3) F(ab′)₂, (4) Fv, and (5) SCA. A specific exampleof “a biologically active antibody fragment” is a CDR region of theantibody. Methods of making these fragments are known in the art, seefor example, Harlow and Lane (1988) Supra.

The isolation of other hybridomas secreting monoclonal antibodies withthe specificity of the monoclonal antibodies of the invention can alsobe accomplished by one of ordinary skill in the art by producinganti-idiotypic antibodies (Herlyn, et al. (1986) Science 232: 100). Ananti-idiotypic antibody is an antibody which recognizes uniquedeterminants present on the monoclonal antibody produced by thehybridoma of interest.

Idiotypic identity between monoclonal antibodies of two hybridomasdemonstrates that the two monoclonal antibodies are the same withrespect to their recognition of the same epitopic determinant. Thus, byusing antibodies to the epitopic determinants on a monoclonal antibodyit is possible to identify other hybridomas expressing monoclonalantibodies of the same epitopic specificity.

Compositions containing the antibodies, fragments thereof or cell lineswhich produce the antibodies, are encompassed by this invention. Whenthese compositions are to be used pharmaceutically, they can be combinedwith a pharmaceutically acceptable carrier.

Pulsing Antigen Presenting Cells

The polypeptides of this invention also can be pulsed into antigenpresenting cells using the methods described herein either in vivo or invitro. Various methods of pulsing the antigen presenting cells aredisclosed in U.S. Pat. No. 6,306,640, Lodge et al. (2000), Cancer Res.60:829, Lau et al. (2001), J Immun. 24(1):66, Gajewski et al. (2001),Clin. Cancer Res. 7:895s, Morse et al. (1999), Clin. Cancer Res. 5:1331and Schmidt et al. (1997) Proc. Natl. Acad. Sci. 94:3262.Antigen-presenting cells, include, but are not limited to dendriticcells (DCs), monocytes/macrophages, B lymphocytes or other cell type(s)expressing the necessary MHC/co-stimulatory molecules. The methodsdescribed below focus primarily on DCs which are the most potent,preferred APCs. These host cells containing the polypeptides or proteinsare further provided.

The terms “antigen-presenting cells” or “APCs” includes both intact,whole cells as well as other molecules which are capable of inducing thepresentation of one or more antigens, preferably in association with MHCmolecules. Examples of suitable APCs are discussed in detail below andinclude, but are not limited to, whole cells such as macrophages,dendritic cells, B cells, purified MHC class I molecules complexed tobeta 2-microglobulin; and foster antigen presenting cells.

Dendritic cells are the most effective type of antigen presenting cells(APC) in the human body expressing significant levels of co-stimulatory(CD86, CD80) and MHC class I and class II molecules on their cellsurface. Many different factors and cytokine combinations have beendemonstrated to produce mature dendritic cells (mDCs) in vitro. It hasbeen shown that DCs provide all the signals required for T cellactivation and proliferation. These signals can be categorized into twotypes. The first type, which gives specificity to the immune response,is mediated through interaction between the T-cell receptor/CD3(“TCR/CD3”) complex and an antigenic peptide presented by a majorhistocompatibility complex (“MHC”) class I or II protein on the surfaceof APCs. This interaction is necessary, but not sufficient, for T cellactivation to occur. In fact, without the second type of signals, thefirst type of signals can result in T cell anergy. The second type ofsignals, called costimulatory signals, is neither antigen-specific norMHC-restricted, and can lead to a full proliferation response of T cellsand induction of T cell effector functions in the presence of the firsttype of signals. As used herein, “dendritic cell” is to include, but notbe limited to a pulsed dendritic cell, a foster cell or a dendritic cellhybrid.

The term “immune effector cells” refers to cells capable of binding anantigen or which mediate an immune response. These cells include, butare not limited to, T cells, B cells, monocytes, macrophages, NK cellsand cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones,and CTLs from tumor, inflammatory, or other infiltrates. A “naive” cellis a cell that has never been exposed to an antigen. As demonstrated inthe Examples, the present methods, peptides and effector cells caninduce or provide not only significant levels of cytotoxicity, butlevels of cytotoxicity from about 20 percent up to about 64 percent, ormore specifically 21, 29, 31, 33, 34, 35, 36, 37 or 39 percent to 42,43, 55, 56, 63 or 64 percent in various cell types.

Isolated host cells which present the polypeptides of this invention inthe context of MHC molecules are further useful to expand and isolate apopulation of educated, antigen-specific immune effector cells. Theimmune effector cells, e.g., cytotoxic T lymphocytes, are preferablyproduced by culturing naive immune effector cells withantigen-presenting cells that present the polypeptides in the context ofMHC molecules on the surface of the APCs. The population can be purifiedusing methods known in the art, e.g., FACS analysis or ficoll gradient.The methods to generate and culture the immune effector cells as well asthe populations produced thereby also are the inventor's contributionand invention. Pharmaceutical compositions comprising the cells andpharmaceutically acceptable carriers are useful in adoptiveimmunotherapy. Prior to administration in vivo, the immune effectorcells can be screened in vitro for their ability to lyse melanoma tumorcells.

In one embodiment, the immune effector cells and/or the APCs aregenetically modified. Using standard gene transfer, genes coding forco-stimulatory molecules and/or stimulatory cytokines can be insertedprior to, concurrent to or subsequent to expansion of the immuneeffector cells.

Immune Effector Cells

The present invention also encompasses these immune effector cells thathave been exposed to polypeptides of the present invention, preferablyin an isolated form. Alternative to the above, the immune effector cellscan be exposed to the polypeptides, preferably in the presence of one ormore stimulatory molecules, without the help of antigen presentingcells.

Immune Response Induction

This invention also provides methods of inducing an immune response in asubject, comprising administering to the subject an effective amount ofthe polypeptides described above under the conditions that induce animmune response to the polypeptide. The polypeptides can be administeredin formulations or as polynucleotides encoding the polypeptides. Thepolynucleotides can be administered in a gene delivery vehicle or byinserting into a host cell which in turn recombinantly transcribes,translates and processes the encoded polypeptide. Isolated host cellscontaining the polynucleotides of this invention in a pharmaceuticallyacceptable carrier can therefore combined with appropriate and effectiveamount of an adjuvant, cytokine or co-stimulatory molecule for aneffective vaccine regimen. The vaccination can either be prophylactic orfor treatment of established cancer. In one embodiment, the host cell isan APC such as a dendritic cell. The host cell can be further modifiedby inserting a polynucleotide coding for an effective amount of eitheror both of a cytokine a co-stimulatory molecule.

The methods of this invention can be further modified byco-administering an effective amount of a cytokine or co-stimulatorymolecule. As used herein, the term “cytokine” refers to any one of thenumerous factors that exert a variety of effects on cells, for example,inducing growth or proliferation. Non-limiting examples of cytokineswhich can be used alone or in combination in the practice of the presentinvention include, interleukin-2 (IL-2), stem cell factor (SCF),interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 12 (IL-12),G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF),interleukin-1 alpha (IL-1.sub.I), interleukin-11 (IL-11), MIP-1I,leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO) andflt3 ligand. The present invention also includes culture conditions inwhich one or more cytokine is specifically excluded from the medium.Cytokines are commercially available from several vendors such as, forexample, Genzyme (Framingham, Mass.), Genentech (South San Francisco,Calif.), Amgen (Thousand Oaks, Calif.), R&D Systems and Immunex(Seattle, Wash.). It is intended, although not always explicitly stated,that molecules having similar biological activity as wild-type orpurified cytokines (e.g., recombinantly produced or muteins thereof) areintended to be used within the spirit and scope of the invention.

“Co-stimulatory molecules” are involved in the interaction betweenreceptor-ligand pairs expressed on the surface of antigen presentingcells and T cells. One exemplary receptor-ligand pair is the B7co-stimulatory molecules on the surface of DCs and its counter-receptorCD28 or CTLA-4 on T cells (Freeman, et al. (1993) Science 262:909-91 1;Young, et al. (1992) J. Clin. Invest. 90: 229; Nabavi, et al. Nature360:266). Other important co-stimulatory molecules are CD40, CD54, CD80,CD86.

Patient T cell assays can generally be performed by treating patientPBMCs with the reactive antigens and analyzing the cells for a suitableresponse. For example, the PBMC supernatant can be assayed for the levelof secreted cytokines. Preferably, the cytokine assayed isinterferon-gamma, interleukin-2, interleukin-12 (either the p40 subunitor biologically active p70), interleukin-1 or tumor necrosis factor-α.The cytokines interleukin-4 and interleukin-10 can also be assayed,since the levels of these representative Th2-type cytokines generallydecrease in response to treatment with a polypeptide as describedherein. Cytokines can be assayed, for example, using commerciallyavailable antibodies specific for the cytokine of interest in an ELISAformat, with positive results determined according to the manufacturer'sinstructions. Suitable antibodies can be obtained, for example, fromChemicon, Temucula, Calif. and PharMingen, San Diego, Calif.Alternatively, the treated PBMCs can be assayed for mRNA encoding one ormore of the cytokines interferon-gamma, interleukin-2, interleukin-12p40 subunit, interleukin-1 or tumor necrosis factor-α, or the PBMCs canbe assayed for a proliferative response as described herein.Alternatively, cytokines can be measured by testing PBMC supernatantsfor cytokine-specific biological activities.

Method of Diagnosis

According to one aspect of the invention, methods for diagnosing adisorder that is characterized by expression of a leukemia associatednucleic acid or polypeptide are provided. The methods involve contactinga biological sample isolated from a subject with an agent specific forthe leukemia associated nucleic acid or polypeptide to detect thepresence of the leukemia associated nucleic acid or polypeptide in thebiological sample. As used herein, “contacting” means placing thebiological sample in sufficient proximity to the agent and under theappropriate conditions of, e.g., concentration, temperature, time, ionicstrength, to allow the specific interaction between the agent andleukemia associated nucleic acid or polypeptide that are present in thebiological sample. In general, the conditions for contacting the agentwith the biological sample are conditions known by those of ordinaryskill in the art to facilitate a specific interaction between a moleculeand its cognate (e.g., a protein and its receptor cognate, an antibodyand its protein antigen cognate, a nucleic acid and its complementarysequence cognate) in a biological sample. Exemplary conditions forfacilitating a specific interaction between a molecule and its cognateare described in U.S. Pat. No. 5,108,921, issued to Low et al.

The biological sample can be located in vivo or in vitro. For example,the biological sample can be a hematopoietic tissue in vivo and theagent specific for the leukemia associated nucleic acid or polypeptidecan be used to detect the presence of such molecules in thehematopoietic tissue (e.g., for imaging portions of the hematopoietictissue that express the leukemia associated gene products).Alternatively, the biological sample can be located in vitro (e.g., ablood sample, bone marrow biopsy, tissue extract). In a particularlypreferred embodiment, the biological sample can be a cell-containingsample, more preferably a sample containing hematopoietic cells.

The skilled artisan can determine which HLA molecule binds to the CD33fragments by, e.g., experiments utilizing antibodies to blockspecifically individual HLA class I molecules. For example, antibodieswhich bind selectively to HLA-A2 will prevent efficient presentation ofantigens specifically presented by HLA-A2. Thus, if the present peptidesare presented by HLA-A2, then the inclusion of anti-HLA-A2 antibodies inan in vitro assay will block the presentation of these antigens. Anassay for determining the nature of the HLA molecule is found in U.S.Pat. No. 5,939,526.

Vaccine

The present invention also provides vaccine compositions comprising theCD33 antigen peptide fragments or nucleic acids encoding these fragmentsdescribed above. Vaccines can also be prepared from antigen presentingcells that have been pulsed with the peptides or nucleic acids or immuneeffector cells which have been exposed to the peptides or nucleic acids.The vaccine can contain a single peptide or a range of peptides whichcover different or similar epitopes. In addition or alternatively, thevaccine can be a polyvalent vaccine where a single polypeptide can beprovided with multiple epitopes.

In one embodiment the peptide is conjugated to a carrier protein, suchas for example a polycation (poly-L-Lysine or poly-L-arginine), tetanustoxoid, diphtheria toxoid or oxidised KLH in order to stimulate T cellhelp as disclosed in U.S. Pat. No. 6,344,203.

Included as part of the vaccine, substances which potentiate the immuneresponse can be administered with nucleic acid or peptide components ina cancer vaccine. Such immune response potentiating compounds can beclassified as either adjuvants or cytokines. Adjuvants can enhance theimmunological response by providing a reservoir of antigen(extracellularly or within macrophages), activating macrophages andstimulating specific sets of lymphocytes. Adjuvants of many kinds arewell known in the art; specific examples include MPL (SmithKlineBeecham), a congener obtained after purification and acid hydrolysis ofSalmonella minnesota Re 595 lipopolysaccharide, QS21 (SmithKlineBeecham), a pure QA-21 saponin purified from Quillija saponaria extract,and various water-in-oil emulsions prepared from biodegradable oils suchas squalene and/or tocopherol. Cytokines are also useful in vaccinationprotocols as a result of lymphocyte stimulatory properties. Manycytokines useful for such purposes will be known to one of ordinaryskill in the art, including interleukin-12 (IL-12) which has been shownto enhance the protective effects of vaccines (Science 268: 1432-1434,1995).

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations can routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents.

The term “pharmaceutically acceptable” means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredients. The term “physiologically acceptable” refers toa non-toxic material that is compatible with a biological system such asa cell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. Initial doses can also be followed by booster doses, followingimmunization protocols standard in the art. The administration may, forexample, be oral, intravenous, intraperitoneal, intramuscular,intracavity, subcutaneous, or transdermal. When antibodies are usedtherapeutically, a preferred route of administration is by pulmonaryaerosol. Techniques for preparing aerosol delivery systems containingantibodies are well known to those of skill in the art. Generally, suchsystems should utilize components which will not significantly impairthe biological properties of the antibodies, such as the paratopebinding capacity (see, for example, Sciarra and Cutie, “Aerosols,” inRemington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712).Those of skill in the art can readily determine the various parametersand conditions for producing antibody aerosols without resort to undueexperimentation. When using antisense preparations of the invention,slow intravenous administration is preferred.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

In all cases where cells are used as a vaccine, these can be cellstransfected with coding sequences for one or both of the componentsnecessary to provoke a CTL response, or be cells which already expressboth molecules without the need for transfection. Vaccines alsoencompass naked DNA or RNA, encoding the present peptides, which can beproduced in vitro and administered via injection, particle bombardment,nasal aspiration and other methods. Vaccines of the “naked nucleic acid”type have been demonstrated to provoke an immunological responseincluding generation of CTLs specific for the peptide encoded by thenaked nucleic acid (Science 259:1745-1748, 1993). When “disorder” isused herein, it refers to any pathological condition where the tumorrejection antigen precursor is expressed. An example of such a disorderis cancer, leukemias and lymphomas in particular.

The peptides of the present invention can also be used to elicit orenhance an immune response to an antigen encoded by a DNA vaccine. DNAvaccines encode one or more immunostimulating antigens, such that theantigen is generated in situ. For instance, the DNA vaccine can encode atumor antigen and, optionally, a peptide as described herein. In suchvaccines, the DNA can be present within any of a variety of deliverysystems known to those of ordinary skill in the art, including nucleicacid expression systems, bacteria and viral expression systems.Appropriate nucleic acid expression systems contain the necessary DNAsequences for expression in the patient (such as a suitable promoter).Bacterial delivery systems involve the administration of a bacterium(such as Bacillus-Calmette-Guerrin) that expresses an epitope of aleukemia cell antigen on its cell surface. The DNA can be introducedusing a viral expression system (e.g., vaccinia or other pox virus,retrovirus, or adenovirus), which can involve the use of anon-pathogenic (defective), replication competent virus. Suitablesystems are disclosed, for example, in Fisher-Hoch et al., PNAS86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103, 1989;Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112,4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627,1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., PNAS91:215-219, 1994; Kass-Eisler et al., PNAS 90:11498-11502, 1993; Guzmanet al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res.73:1202-1207, 1993. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.The DNA can also be “naked,” as described, for example, in published PCTapplication WO 90/11092, and Ulmer et al., Science 259:1745-1749, 1993,reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNAcan be increased by coating the DNA onto biodegradable beads, which areefficiently transported into the cells.

Method of Treatment

The present invention provides a method of treating individualssuffering from leukemia. In such methods, the introduction of peptides,nucleic acids, protein binding agents, antigen presenting cells and/orimmune effector cells as described above serves as an immunotherapeutic,directing and promoting the immune system of the individual to combatleukemic cells that display the CD33 antigen fragments. The methods cancomprise administering an effective amount of any of the above compoundsto a patient in need of such treatment through the means describedabove. The methods can further comprise a course of chemotherapy, suchas with 5-FU or cisplatin, prior to administration of the abovecompounds. See, e.g. Int. J. Cancer; 101:265 (2002).

Individuals at risk of developing leukemia, such as those having agenetic predisposition, can be treated with the formulations of thepresent in a prophylactic attempt to delay or eliminate the onset of theleukemic state. Similarly, those individuals who have already developedcancer and who have been treated to remove the cancer or are otherwisein remission are particularly susceptible to relapse and reoccurrence.As part of a treatment regimen, such individuals can be immunizedagainst the cancer that they have been diagnosed as having had in orderto combat a recurrence. Thus, once it is known that an individual hashad a type of cancer and is at risk of a relapse, they can be immunizedin order to prepare their immune system to combat any future appearanceof the cancer.

Therapeutic approaches based upon the disclosure are premised on aresponse by a subject's immune system, potentially leading to lysis ofleukemia cells. One such approach is the administration of autologousCTLs specific to the complex to a subject with abnormal cells of thephenotype at issue. It is within the skill of the artisan to developsuch CTLs in vitro. Generally, a sample of cells taken from a subject,such as blood cells, are contacted with a cell presenting the complexand capable of provoking CTLs to proliferate. The target cell can be atransfectant. These transfectants present the desired complex of theirsurface and, when combined with a CTL of interest, stimulate itsproliferation. Specific production of a CTL is well known to one ofordinary skill in the art. The clonally expanded autologous CTLs canthen be administered to the subject.

In one therapeutic methodology, referred to as adoptive transfer(Greenberg, J. Immunol. 136(5): 1917, 1986; Riddel et al., Science 257:238, 1992; Lynch et al, Eur. J. Immunol. 21: 1403-1410, 1991; Kast etal., Cell 59: 603-614, 1989), cells presenting the desired complex arecombined with CTLs leading to proliferation of the CTLs specificthereto. The proliferated CTLs are then administered to a subject with acellular abnormality which is characterized by certain of the abnormalcells presenting the particular complex. The CTLs can then lyse theabnormal cells, thereby achieving the desired therapeutic goal.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present the relevant HLA complex. This can be determinedvery easily, as the art is very familiar with methods for identifyingcells which present a particular HLA molecule, as well as how toidentify cells expressing DNA of the pertinent sequences, in this case aleukemia associated gene sequence. Once cells presenting the relevantcomplex are identified via the foregoing screening methodology, they canbe combined with a sample from a patient, where the sample containsCTLs. If the complex presenting cells are lysed by the mixed CTL sample,then it can be assumed that a leukemia associated gene is beingpresented, and the subject is an appropriate candidate for thetherapeutic approaches set forth herein.

Adoptive transfer is not the only form of therapy that is available inaccordance with the invention. CTLs can also be provoked in vivo, usinga number of approaches. One approach is the use of non-proliferativecells expressing the complex, such as antigen presenting cells. Thecells used in this approach can be those that normally express thecomplex, such as irradiated tumor cells or cells transfected with one orboth of the genes necessary for presentation of the complex. Chen etal., Proc. Natl. Acad. Sci. USA 88: 110-114 (1991) exemplifies thisapproach, showing the use of transfected cells expressing HPV E7peptides in a therapeutic regime. Various cell types can be used.Similarly, vectors carrying one or both of the genes of interest can beused. Viral or bacterial vectors are especially preferred. The nucleicacid can be incorporated into an expression vector. Expression vectorscan be unmodified extrachromosomal nucleic acids, plasmids or viralgenomes constructed or modified to enable insertion of exogenous nucleicacids, such as those encoding the present peptides. Nucleic acidsencoding these peptides can also be inserted into a retroviral genome,thereby facilitating integration of the nucleic acid into the genome ofthe target tissue or cell type. In these systems, the gene of interestis carried by a microorganism, e.g., a Vaccinia virus, retrovirus or thebacteria BCG, and the materials defacto “infect” host cells. The cellswhich result present the complex of interest, and are recognized byautologous CTLs, which then proliferate.

Kits

The invention also provides isolated proteins and peptides, andantibodies to those proteins and peptides. Kits containing any of theforegoing molecules, alone or in combination, are additionally provided.The foregoing can be used in the diagnosis or treatment of conditionscharacterized by the expression of the present peptides. The kits canalso be used to pulse antigen presenting cells or t-lymphocytes, and, assuch, can contain appropriate culture media, culture media supplementssuch as cytokines, disposable laboratory equipment and the like.Examples of such kit components can be found in the following examples.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepharmaceutical compositions can be employed in conjunction with othertherapeutic compounds.

The invention in another aspect involves a kit for detecting thepresence of the expression of the present polypeptide. Such kits employtwo or more of the above-described nucleic acid molecules isolated inseparate containers and packaged in a single package. In one such kit, apair of isolated nucleic acid molecules is provided. In certainembodiments, the pair of isolated nucleic acid molecules are PCRprimers.

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components can be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

This invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1

A total of 364 amino acids of CD33 protein, listed above as SEQ ID NO:4, were retrived from the SWISS-PROT databank and analyzed for HLA-A2.1binding epitopes as nonamers by the peptide motif search softwareSYFPEITHI which is supported by DFG-Sonderforschungsbereich 510 and theEuropean Union: EU BIOMED CT95-1627, BIOTECH CT95-0263, and EUQLQ-CT-1999-00713. The algorithms used are based on the book “MHCLigands and Peptide Motifs” by H. G. Rammensee, J. Bachmann and S.Stevanovic. The prediction of the SYFPEITHI program is based onpublished motifs (pool sequencing, natural ligands) and the score iscalculated by the consideration of specific amino acids of the peptidein a numeric value depending on whether they are carrying anchor,auxiliary anchor, or preferred residue. Ideal anchors are given 10points, unusual anchors 6-8 points, auxiliary anchors 4-6 points andpreferred residues 1-4 points. Amino acids that are regarded as having anegative effect on the binding are given values between −1 and −3.Besides a prediction for binding, the preferred amino acids in thepeptide of CD33 antigen were examined for the possible immunogenicepitopes.

Based on high HLA-A2.1 binding scores, several 9 mer fragments of theCD33 peptide SEQ ID NO: 4) were identified using SYFPEITHI software andare listed below. The score means the calculated potential capabilityfor binding to a HLA-A2 molecule.

SEQ Position 1 2 3 4 5 6 7 8 9 Score ID NO: 269 A L L A L C L C L 26 8270 L L A L C L C L I 26 42 2 P L L L L L P L L 25 43 4 L L L L P L L WA 24 44 65 A I I S G D S P V 24 2 133 Q L S V H V T D L 24 45 276 C L IF F I V K T 24 46 147 I L I P G T L E P 23 47 5 L L L P L L W A G 22 4830 T V Q E G L C V L 22 49 179 W L S A A P T S L 22 50 226 T I Q L N V TY V 22 9 9 L L W A G A L A M 21 51 34 G L C V L V P C T 21 52 6 L L P LL W A G A 20 53 74 A T N K L D Q E V 20 54 261 A I G G A G V T A 20 55274 C L C L I F F I V 20 56 15 L A M D P N F W L 19 57 262 I G G A G V TA L 19 58 263 G G A G V T A L L 19 59 267 V T A L L A L C L 19 60 1 M PL L L L L P L 18 61 221 V T T E R T I Q L 18 62 265 A G V T A L L A L 1863 315 K L H G P T E T S 18 64 66 I I S G D S P V A 17 65 77 K L D Q E VQ E E 17 66 140 D L T H R P K I L 17 67 160 N L T C S V S W A 17 68 172G T P P I F S W L 17 69 191 T T H S S V L I I 17 70 259 H G A I G G A GV 17 71 273 L C L C L I F F I 17 72 324 S C S G A A P T V 17 73 3 L L LL L P L L W 16 74 23 L Q V Q E S V T V 16 75 29 V T V Q E G L C V 16 76148 L I P G T L E P G 16 77 186 S L G P R T T H S 16 78 209 N L T C Q VK F A 16 79 241 G I F P G D G S G 16 80 249 G K Q E T R A G V 16 81 257V V H G A I G G A 16 82 272 A L C L C L I F F 16 83 333 E M D E E L H YA 16 84 7 L P L L W A G A L 15 85 8 P L L W A G A L A 15 86 14 A L A M DP N F W 15 87 21 F W L Q V Q E S V 15 88 31 V Q E G L C V L V 15 89 37 VL V P C T F F H 15 90 98 R N N C S L S I V 15 91 128 S Y K S P Q L S V15 92 141 L T H R P K I L I 15 93 152 T L E P G H S K N 15 94 175 P I FS W L S A A 15 95 213 Q V K F A G A G V 15 96 260 G A I G G A G V T 1597 277 L I F F I V K T H 15 98 326 S G A A P T V E M 15 99 330 P T V E MD E E L 15 100 353 D T S T E Y S E V 15 101 22 W L Q V Q E S V T 14 10270 D S P V A T N K L 14 103 91 R L L G D P S R N 14 104 92 L L G D P S RN N 14 105 97 S R N N C S L S I 14 106 102 S L S I V D A R R 14 107 130K S P Q L S V H V 14 108 157 H S K N L T C S V 14 109 168 A C E Q G T PP I 14 110 197 L I I T P R P Q D 14 111 253 T R A G V V H G A 14 112 347G M N P S K D T S 14 113 59 Y W F R E G A I I 13 114 85 E T Q G R F R LL 13 115 139 T D L T H R P K I 13 116 151 G T L E P G H S K 13 117 153 LE P G H S K N L 13 118 188 G P R T T H S S V 13 119 190 R T T H S S V LI 13 120 196 V L I I T P R P Q 13 121 215 K F A G A G V T T 13 122 216 FA G A G V T T E 13 123 228 Q L N V T Y V P Q 13 124 234 V P Q N P T T GI 13 125 254 R A G V V H G A I 13 126 291 R T A V G R N D T 13 127 298 DT H P T T G S A 13 128 327 G A A P T V E M D 13 129 10 L W A G A L A M D12 130 16 A M D P N F W L Q 12 131 17 M D P N F W L Q V 12 132 126 K Y SY K S P Q L 12 133 146 K I L I P G T L E 12 134 181 S A A P T S L G P 12135 206 H G T N L T C Q V 12 136 219 A G V T T E R T I 12 137 231 V T YV P Q N P T 12 138 314 S K L H G P T E T 12 139 335 D E E L H Y A S L 12140 340 Y A S L N F H G M 12 141 342 S L N F H G M N P 12 142 39 V P C TF F H P I 11 143 68 S G D S P V A T N 11 144 95 D P S R N N C S L 11 145104 S I V D A R R R D 11 146 119 R M E R G S T K Y 11 147 131 S P Q L SV H V T 11 148 145 P K I L I P G T L 11 149 195 S V L I I T P R P 11 150198 I I T P R P Q D H 11 151 202 R P Q D H G T N L 11 152 218 G A G V TT E R T 11 153 223 T E R T I Q L N V 11 154 225 R T I Q L N V T Y 11 155266 G V T A L L A L C 11 156 281 I V K T H R R K A 11 157 305 S A S P KH Q K K 11 158 24 Q V Q E S V T V Q 10 159 48 P Y Y D K N S P V 10 16058 G Y W F R E G A I 10 161 67 I S G D S P V A T 10 162 78 L D Q E V Q EE T 10 163 100 N C S L S I V D A 10 164 144 R P K I L I P G T 10 165 182A A P T S L G P R 10 166 217 A G A G V T T E R 10 167 250 K Q E T R A GV V 10 168 264 G A G V T A L L A 10 169 268 T A L L A L C L C 10 170 271L A L C L C L I F 10 171 286 R R K A A R T A V 10 172 288 K A A R T A VG R 10 173 289 A A R T A V G R N 10 174 295 G R N D T H P T T 10 175 308P K H Q K K S K L 10 176 323 S S C S G A A P T 10 177 355 S T E Y S E VR T 10 178

The following peptides were tested for their ability to bind toHLA-A2.1+ T2 cells and their binding scores under Brefeldin A treatment.Eur. J. Immunol. 2000.30:3411-3421. This experiment was performed tomeasure the peptide/HLA-A2.1 complex stability using the native ormodified CD33 peptide. As the immunogenicity of peptides dependsprimarily on their capacity to stabilize the HLA-A2.1 molecules, morestable peptide to HLA-A2.1 could generate more effective cytotoxic Tcells to leukemia cells.

Brefeldin A treatment Peptide Sequence None 0 hr 2 hr 4 hr 6 hrOvernight T2 alone 215 226 200 187 157 255 SEQ ID NO: 42 LLALCLCLI 204304 280 233 213 193 SEQ ID NO: 2 AIISGDSPV 566 647 332 188 142 428 SEQID NO: 3 YIISGDSPV 547 641 588 380 216 248 SEQ ID NO: 10 YIGSGDSPV 515687 285 167 151 280 SEQ ID NO: 11 YIIIGDSPV 263 301 257 208 172 325 SEQID NO: 12 YIILGDSPV 465 488 315 205 173 323 SEQ ID NO: 6 YIISGISPV 591840 929 839 680 593 SEQ ID NO: 13 YIISGDLPV 619 724 524 307 203 296 SEQID NO: 14 YIISGDSWV 209 247 247 208 173 298 SEQ ID NO: 15 YIISGDSPL 428426 334 217 168 157 SEQ ID NO: 7 YLISGDSPV 701 728 810 745 594 479 SEQID NO: 16 ALISGDSPV 603 677 882 692 609 366 SEQ ID NO: 9 TIQLNVTYV 175218 173 180 159 404

As can be seen from these results, the peptides of SEQ ID NOS: 6, 7 and16 gave good and prolonged stable binding. Several other peptidesprovided good initial binding over baseline values.

Example 2

The present example illustrates that that the present peptides arecapable of inducing a T-lymphocyte response. In this example, antigenpresenting cells were pulsed at 150 micrograms of each peptide per 1million antigen presenting cells with the following amino acidsequences:

-   -   (1) YLALCLCLI (SEQ ID NO: 1);    -   (2) AIISGDSPV (SEQ ID NO: 2); and    -   (3) YIISGDSPV (SEQ ID NO: 3).

Other peptides that include the sequences listed above can be similarlytested by the skilled artisan in the course of normal experimentation todetermine whether peptides including any of the recited sequences can beeffectively used as described herein.

The antigen presenting cells were then contacted with effector cells ata target effector ration of 1:60. The effector cells were then contactedwith HLA-A2.1 positive ML-2 cells and the cytotoxicity to the ML-2 cellswere measured. ML-2 cells are acute myeloid leukemia cells (AML) cellswhich are HLA-A2.1 positive. The results are set forth in Table 1.

TABLE 1 Cytoxicity Cytotoxicity CD33 to ML-1 to AML Antigen PeptideCells by Patient's Experiment Presenting (SEQ ID Number of CytokinesBone Number Cells Donor NO:) Stimulations (%) Marrow (%) Control None B,F, G None 0 0, 0, 7 1 DC A 2 2 37 2 DC B 2 2 42 43 3 DC C 2 2 39 63 4 DCD 2 2 29 5 DC E 3 2 37 6 T2 F 1 2 3 21 36 7 T2 G 1 2 3 34 64 8 T2 F 2 23 33 56 9 T2 G 2 2 3 35 55

Table 2 shows the expression levels of different types of effector cellscontacted with antigen presenting cells pulsed with examples of peptidesof the present invention. Results are shown in mean fluorescenceintensity. Peptides inducing 20% or greater cytotoxicity are generallyconsidered to promote significant cytotoxicity. Testing the cytotoxicitylevel of an effector cell exposed to a peptide is a major method formeasuring the effectiveness for the peptide.

TABLE 2 CD33 Antigen Peptide Expression levels of: Presenting (SEQ IDNumber CD45RO CD95 CD4 CD8 Cells Donor NO:) of Stims Memory Activation Thelper T cytotoxic None F, G None 0  420  24 120 2430 DC C 2 2 1851 110332 2477 DC D 2 2 2593 172 356 2332 T2 F 2 2 3 2146 1963  77 127 51 496267 7793 T2 G 2 2 3 1575 1591 133 152 31 36 8070 8187 T2 F 1 2 1262  99 81 3381 T2 G 1 2 1432 102  57 7796

FIG. 1A shows IFN-γ release by cytotoxic T-lymphocytes (CTLs) from twodifferent HLA-A2.1+ (shown as diamond or box) donors stimulated bydendritic cells pulsed three times, once a week with the AIISGDSPV (SEQID NO:2) peptide. FIG. 1B shows IFN-γ released by CTLs stimulated by T2cells pulsed as above with the AIISGDSPV (SEQ ID NO:2) peptide. As canbe seen from these FIGS., the highest IFN-γ release was observed whenthe CTLs were stimulated twice. IFN-γ amount is shown inpicogram/milliliter. The scale for FIG. 1B is the same as for FIG. 1A.

The following peptide has also been determined to be immunogenic insimilar experiments, ALISGDSPV (SEQ ID NO: 16).

Example 3

The search software SYFPEITHI was used to identify the potential peptidemotifs to HLA-A2.1-specific CD33 epitopes. Among the peptides predicted,four peptides were selected and synthesized (>95% pure) for evaluationbased on high scores for HLA-A2.1 binding and the presence of primaryHLA-A2.1 anchor residues. T2 cells, a transporter antigen processing(TAP) gene-deficient cell line which express only HLA-A2.1 MHC class Imolecules (Zweerink et al., 1993), were used to evaluate CD33 peptidespecific binding to HLA-A2.1 on the cells. Influenza virus matrixpeptide 58-66 (GILGFVFTL SEQ ID NO: 179), which is known to bind verytightly to HLA-A2.1, was used as an HLA-A2.1-specific control peptide.

Peptide Synthesis

Four potential HLA-A2.1-specific CD33 epitopes: LLALCLCLI (residues270-278, SEQ ID NO: 42), ALLALCLCL (residues 269-277, SEQ ID NO: 8),AIISGDSPV (residues 65-73, SEQ ID NO: 2) and TIQLNVTYV (residues226-234, SEQ ID NO:9) were synthesized by standard fmoc(9-fluorenylmethyl-oxycarbonyl) chemistries and were purified to >90%using reverse-phase chromatography (Biosynthesis, Lewisville, Tex.). AHLA-A2.1-specific influenza virus protein matrix peptide (GILGFVFTL:residues 58-66, SEQ ID NO: 179) was synthesized and used as a positivecontrol in these studies. The identity of each peptide was validated bymass-spectrometry for molecular weight.

Cell Lines

AML cell lines, EOL-1 (DSMZ, Braunschweig, Germany), Kasumi-1 (DSMZ),HL-60 (ATCC, Manassas, Va.), SR-91 (ATCC), THP-1 (ATCC), ML-2 (gift fromDr. Y. Matsuo, Okayama, Japan) were maintained in liquid culture in RPMI1640 and 10% fetal calf serum (FCS; Biowhittaker, Walkersville, Md.). Inaddition, the AML cell lines, UT-2 (alpha-MEM, 20% FCS, 5 ng/ml GM-CSF),OCI-AML5 (alpha-MEM, 20% FCS, 10 ng/ml GM-CSF), and MUTZ-2 (alpha-MEM,20% FCS, 50 ng/ml SCF) were purchased from DSMZ (Braunschweig, Germany).T2 cells, a human B and T cell hybrid expressing HLA-A2.1, weremaintained in RPMI 1640 plus 20% FCS and used as antigen presentingcells in these studies.

Reagents

Monoclonal mouse anti-HLA-A2.1 antibody was purified from the culturesupernatant of the hybridoma BB7.2 cell line (ATCC). Recombinant humanGM-CSF and CD40 ligand were obtained from Immunex (Seattle, Wash.).Recombinant human IL-2, IL-4, IL-6, IL-7, IFN-α, IFN-γ, TNF-α, and IL-12were purchased from R&D Systems (Minneapolis, Minn.). Mouse anti-humanmonoclonal antibodies (CD40, CD69, CD80, CD83, and CD86) conjugated witheither FITC or PE were purchased from Immunotech (Hialeigha, Fla.), andeither fluorecently labeled or purified antibodies to CD3, CD4, CD8,CD14, CD28, CD33, CD45RA, CD45RO and HLA-DR were purchased from BectonDickinson Pharmingen (San Diego, Calif.). Purified lipopolysacchride(LPS) prepared from Escherichia coli O:113 was obtained from theNational Institute of Health (Bethesda, Md.).

MHC Peptide Binding Assay

The assay for peptide binding to HLA-A2.1 was performed (Nijman et al.,1993) using the TAP-deficient T2 hybrid cell line, which is known toup-regulate HLA-A2.1 expression on the cell surface by acquiring onlyexogenous epitope (Salter et al., 1986; Zweerink et al., 1993). T2 cellswere washed and resuspended in serum-free AIM-V™ (Gibco-LifeTechnologies, Rockville, Md.) at a final concentration of 1×10⁶ cells/mland transferred into a 24-well tissue culture plate. Cells were pulsedwith respective CD33 peptides at different concentrations (5-150 μg/ml)or influenza virus protein matrix peptide (30 μg/ml) plus 3 μg humanβ2-microglobulin (Sigma), and incubated for overnight at 37° C., 5% CO₂in humidified air. After incubation, cells were washed once with PBScontaining 3% FCS, and stained with mouse anti-HLA-A2.1 monoclonalantibody for 15 minutes at 4° C. After washing, the cells were incubatedwith goat anti-mouse IgG (F(ab′)₂)-FITC for 15 minutes at 4° C. Thecells were washed once, and fluorescence was measured on a FACSort™ flowcytometer (Becton Dickson, San Jose, Calif.). The fluorescence index wascalculated as follows: (mean channel fluorescence of sample−mean channelfluorescence of unstained control cells)/mean channel fluorescence ofunstained control cells.

Cell Isolation

Peripheral blood mononuclear cells (PBMCs) were isolated fromheparinized whole blood of healthy HLA-A2.1+ donors by standard gradientcentrifugation with Ficoll-Paque™ Plus (Amersham Pharmacia Biotech AB,Uppsala Sweden). PBMCs were harvested from the interface, washed twice,and resuspended in PBS supplemented with 5 mM EDTA and 0.5% human serumalbumin. Informed consent was obtained from all donors and the protocolwas approved by the Rush Medical School Institutional Review Board.

Monocytes. CD14+ monocytes were separated from PBMCs using a magneticsorting technique (Miltenyi Biotec, Auburn, Calif.). PBMCs wereincubated with colloidal super-paramagnetic microbeads conjugated withanti-human CD14 mAb for 15 minuates at 4° C., and passed over a columnin a magnetic field. After washing, positively enriched CD14+ cells wereeluted from the magnetic columns. Purity (mean±standard deviation) of CD14+ monocytes was examined by flow cytometry and was found to be 92+4%.

T cells. CD3+ T cells were isolated from the monocyte depleted cellfractions using the Pan T cell isolation kit from Miltenyi Biotec. The Tcell isolation was done by depletion of B cells, NK cells, earlyerythroid cells, platelets and basophils by indirectly labeling with acocktail of hapten-conjugated CD11b, CD16, CD19, CD36 and CD56antibodies, and MACs microbeads coupled to an anti-hapten monoclonalantibody. The effluent (negative fraction cells) was collected from thecolumn as the enriched CD3+ T cell fraction. Purity (mean standarddeviation) of CD3+ T cells was examined by flow cytometry and was foundto be 94+4%.

Dendritic Cell (DC) Generation

Immature DCs were generated according to modified protocols of Romani etal. (1994) and Bakker et al. (1995). Briefly, fresh or frozen/thawedCD14+ cells were cultured in RPMI 1640 medium (Gibco-Life Technologies)supplemented with 10% FCS, 1,000 U/ml GM-CSF and 1,000 U/ml IL-4. Thecell cultures were fed with fresh medium and GM-CSF and IL-4 every otherday and cell differentiation was monitored by light microscopy. On day7, the cultures were supplemented with different combinations of DCmaturation factors such as lipopolysaccharide (100 U/ml), TNF-α (10ng/ml), or IFN-α (1,000 U/ml) plus TNF-α (10 ng/ml). After three days ofincubation, mature DCs (mDC) were harvested and evaluated for theirphenotypes by flow cytometry. The maturation factor(s) yielding optimalDC maturation was determined and used to generate mDCs for peptidepulsing in upcoming studies.

Induction of Peptide-Specific CTLs

Two different types of antigen-presenting cells (APCs), mDCs and T2cells, were used to generate CD33 peptide-specific CTLs. APCs werewashed three times in serum-free AIM-V™ culture media and pulsed withpeptide at 150 μg/ml for overnight in the media. The peptide-loaded APCswere then irradiated at 10 Gy, washed once, and resuspended in RPMI 1640media supplemented with 10% human AB serum (Biowhittaker, Walkersville,Md.). Peptide-pulsed APCs were used to prime autologous CD3+ T cells ata 1:20 stimulator-to-responder cell ratio in RPMI 1640 mediasupplemented with 10% human AB serum, 5 ng/ml IL-6, 20 ng/ml IL-7, and 1ng/ml IL-12. CTL cultures were restimulated weekly for a total of 3cycles of stimulation. IL-2 (50 U/ml) was added to the culture one dayafter the second stimulation and the cells were fed three times a weekwith fresh medium containing the cytokines.

Cytotoxicity Asay

The cytolytic activity of the CD33 peptide-specific CTLs was measured ina standard ⁵¹Cr-release assay. The CTLs (effector cells) were seededwith ⁵¹Cr-labeled 5×10³ AML cells (target cells) per well at variouseffector:target cell ratios in 96-well U-bottom microtiter plates andincubated for 4 hours at 37° C., 5% CO₂. The ⁵¹Cr-release was measuredin 100 μl supernatant using a Beckman LS6500 liquid scintillationcounter (Beckman Coulter, Brea, Calif.). The percent specific cell lysiswas calculated as [(experimental release−spontaneous release)÷(maximumrelease−spontaneous release)]. Maximum release was determined fromdetergent-releasable target cell counts and spontaneous release was thetarget cell counts determined in the absence of CTLs.

Cold Target Inhibition Assays

Antigen-specific lysis was evaluated in a cold target inhibition assayby analyzing the capacity of unlabeled AML cells (ML-2) to block lysisof ⁵¹Cr-labeled AML cells. Effector cells were incubated with an equalnumber of the unlabeled “cold” target cells (ML-2) for 1 hour at 37° C.,5% CO₂ before the addition of ⁵¹Cr-labeled “hot” AML target cells. Aftera 4-hour incubation, the supernatants (100 ul) were harvested and thespecific ⁵¹Cr-release was measured. The inhibition of AML-specific lysiswas measured by comparing the percent cytotoxicity of the effector cellsincubated with or without the unlabeled “cold” target cells.

IFN-γ Release by CD33 Peptide-Specific CTLs

IFN-γ release by the CTLs was measured using an IFN-γ ELISA kit(PBL-Biomedical Lab., Piscataway, N.J.). Briefly, IFN-γ standards or thesupernatant from CD33 peptide-specific CTL cultures were transferredinto a 96-well plate pre-coated with anti-human IFN-γ capture monoclonalantibody and incubated for 1 hour in a closed chamber at 24° C. Afterwashing the plate with PBS/0.05% Tween 20, anti-human IFN-γ antibody wasadded to the wells and incubated for 1 hour at 24° C. Wells were thendeveloped by incubation with horseradish peroxidase conjugate and TMBsubstrate solution. Stop solution was added to each well and theabsorbance was determined at 450 nm with a SpectraMAX Plus plate reader(Stratagene, La Jolla, Calif.). The amount of cytokine present in theCTL culture supernatant was calculated based on the IFN-γ standardcurve.

Phenotypic Analysis of CD33 Peptide-Specific CTLs

CTLs were stained with anti-CD8-FITC, -CD45RA-FITC or -CD28-PE,-CD45RO-PE, or -CD69-PE monoclonal antibodies for 15 minutes at 4° C.After incubation, the cells were washed and analyzed by flow cytometry.Live gating of the forward and scatter channels was used to excludedebris and to selectively acquire the lymphocyte population foranalysis. Individual fluorescence data were determined using CellQuest™v2.1 acquisition and analysis software (Becton Dickinson).

Peptide-MHC Tetramer Staining

Streptavidin-PE-labeled HLA-2.1/AIISGDSPV (SEQ ID NO:2) tetramer wasproduced by Beckman Coulter using the methods described by Altman et al.(1996). Two-color flow cytometry assays were performed by stainings withanti-CD8-FITC and tetramer-PE. Briefly, the CTLs (2 ×10⁵ cells) werestained with 300 ng of tetramer and incubated for 30 minutes at 37° C.After a washing, the cells were stained with anti-CD8- FITC mAb for 15minutes at 4° C. Cells were washed and analyzed by flow cytometry.

Results

Identification of HLA-A2.1-Specific CD33 Epitope

The results, expressed as the Fluorescence Index (HLA-A2.1 mean channelfluorescence T2 cells pulsed with β2 microglobulin and CD33 peptide ÷HLA-A2.1 mean channel fluorescence T2 cells pulsed with β2microglobulin) were used to select the best HLA-A2.1 bindingCD33-peptide. A Fluorescence Index (Fl) of > 1.0 indicates the up-regulation of HLA-A2.1 molecules by peptide binding on the surface of T2cells. The peptide AIISGDSPV (SEQ ID NO:2) displayed the highest levelof HLA-A2.1 binding (FI=2.81) (Table 3). Based on these results, thepeptide AIISGDSPV (SEQ ID NO:2) was chosen for evaluation as a potentialimmunogenic epitope to generate CD33-specific CTLs against AML cells.

TABLE 3 Peptide binding assay for HLA-A2.1-specific CD33 peptides CD33Protein Fluorescence Samples Peptide Sequence Domain Index* T2 +β2-microglobulin 1.0 T2 + β2-microglobulin, GILGFVFTL 3.11 pulsed withInfluenza (SEQ ID virus matrix protein NO: 179) peptide₅₈₋₆₆ CD33₂₇₀₋₂₇₈LLALCLCLI Trans- 1.19 (SEQ ID NO: 42) membrane CD33₂₆₉₋₂₇₇ LLALCLCLITrans- 1.04 (SEQ ID NO: 42) membrane CD33₆₅₋₇₃ AIISGDSPV Extracellular2.81 (SEQ ID NO: 42) CD33₂₂₆₋₂₃₄ TIQLNVTYV Extracellular 0.99 (SEQ IDNO: 9) *Fluorescence Index = [HLA-A2.1 mean channel fluorescence T2cells pulsed with CD33 peptide plus β₂ microglobulin ÷ HLA-A2.1 meanchannel fluorescence T2 cells pulsed with β₂ microglobulin only].Fluorescence Index demonstrates the fold increase/decrease of peptidebinding to HLA-A2.1 on the surface of T2 cells and the data representthe results of three separate experiments.Dendritic and T2 Cells as Antigen Presenting Cells

In this example, immature DCs obtained by the culture of CD14+ monocyteswith GM-CSF (1,000 U/ml) and IL-4 (1,000 U/ml) were induced to undergomaturation by incubation with LPS (100 Units/ml), TNF-α 10 ng/ml) orTNF-α (10 ng/ml)+IFN-α 50 ng/ml) during the final three days of theculture period. Flow cytometric analysis of the respective DC culturesshowed a phenotypic profile characterized by high expression of CD40,CD80, CD83, CD86 or HLA-DR and no expression of CD3 or CD14 (data notshown). The highest up-regulation of the co-stimulatory (CD80 and CD86)and HLA-A2.1 MHC class I molecules was detected on DCs treated withTNF-α +IFN-A (Table 5). LPS or TNF-α alone also induced the highexpression of CD80, CD86 and HLA-A2.1 molecules compared to theGM-CSF+IL-4 (immature DC) control group, however the up-regulation wasnot as high as seen treated with the TNF-α+IFN-α combination.

The phenotype of the T2 cell line was evaluated to determine itspotential for use as an alternative type of antigen presenting cell. Theresults (Table 4) showed that T2 cells express high levels ofco-stimulatory and HLA-A2.1 molecules. The expression levels of CD83 andCD86 molecules on T2 cells were comparable to those observed on mDCs(Table 4). The expression of CD80 was higher on T2 cells compared tomDCs. The phenotypic profiles of both the mDCs and T2 cells make themideal candidates for use as antigen presenting cells in the generationof CD33 peptide-specific CTLs.

TABLE 4 Phenotypic analysis of mature dendritic (mDC) and T2antigen-presenting cells. HLA-A2.1 CD80 CD86 CD83 Immature DCs  94  5621  ND¹ DCs matured by LPS 316  71 40 ND DCs matured by TNF-α 475 107 96ND DCs matured by IFN-α + TNF-α 749 137 149  67 T2 cells 577 1214  155 50 ND¹ = not done Phenotypic analysis of culture derived mDC or T2antigen-presenting cells. Immature DCs were generated in vitro fromCD14 + monocytes of HLA-A2.1 + normal donors by incubation with GM-CSFand IL-4 for 10 days in liquid culture. Immature DCs were induced toundergo the maturation by the addition of LPS, TNF- or TNF- + IFN-during the last three days of culture. Results are expressed as the meanchannel fluorescence (MCF) for each antigen tested. Dendritic cellsmatured with TNF- + IFN- displayed the highest levels of HLA-A2.1, CD80,and CD86 expression compared to LPS or TNF- treated dendritic cellcultures. The T2 cell line had the highest level of CD80 expression andsimilar levels of HLA-A2.1 and CD86 expression to the TNF- + IFN-dendritic cells.Cytolytic Activity by CD33 Peptide-Specific CTLs

CD33 peptide-specific CTLs were generated by repeated stimulation ofT-lymphocytes from healthy HLA-A2.1+donors with AIISGDSPV (SEQ IDNO:2)-pulsed autologous mDC. CTLs were harvested one week after thesecond peptide stimulation and examined for their cytolytic activityagainst allogeneic HLA-A2.1+AML patient bone marrow mononuclear cells.Differential counts of the AML patient samples demonstrated a highproportion of blast cells in both AML patient 1 (54% blasts) and patient2 (77% blasts) samples. The CD33-peptide specific CTLs showed 22% and33% of cytolytic activities against bone marrow mononuclear cells fromAML patient 1 and AML patient 2, respectively. Cytokines treatment ofthe AML bone marrow cells with GM-CSF (1,000 U/ml), IFN-γ (100 ng/ml)and TNF-α (15 ng/ml) for 48 hours induced the up-regulation ofco-stimulatory (CD80, CD86) and HLA-A2.1 molecules (FIG. 2), making themmore susceptible to killing by CD33-CTLs. Cytotoxicity results (FIG. 3)showed the enhanced cytolytic activities against cytokines treated-AMLpatients bone marrow mononuclear cells (AML patient 1=43%, AML patient2=63%) compared to the untreated AML cells (AML patient 1=22%, AMLpatient 2=33%).

Autologous mDCs or T2 cells as antigen presenting cells were alsoevaluated to induce AIISGDSPV (SEQ ID NO:2)-specific CTLs. T-lymphocytesfrom healthy HLA-A2.1+ donors were stimulated with either autologous mDCor T2 cells pulsed with the HLA-A2.1-specific AIISGDSPV (SEQ ID NO:2).The CTLs were harvested one week after the second or third stimulationand analyzed for their cytotoxic activities against the ML-2 cells(HLA-A2.1+ AML line). CD33 peptide-specific CTLs generated after twocycles of stimulation via either APC showed comparable cytotoxicities(FIG. 4). Unstimulated control T cells did not display any significantcytotoxicity against the target cells. Also, the data demonstrated thatthe repeated stimulation generated more effective CTLs; stimulationthree times enhanced CTLs activities than two times. Based on thisobservation, generating CTLs with a minimum of three cycles ofstimulation with peptide-pulsed mDCs to obtain highly effective CD33peptide-specific CTLs is preferred. In following experiments, thepossibility of the inhibition of normal cells expressing low levels ofCD33 antigen by the CD33 peptide-specific CTLs was examined. CTLs weregenerated as previously described using mDCs and examined a week aftersecond stimulation. The results indicated no induction of significantcytotoxic activities against PBMCs or monocytes (FIG. 5), suggestingthat CD33 peptide-specific CTLs might not effect the inhibition ofnormal cells.

AML-Specific Cell Lysis

AML specific cell lysis by CD33 peptide-specific CTLs was confirmedusing a cold target inhibition assay. In this assay, pre-incubation ofCD33-CTLs with “cold” ML-2 cells for 1 hour before the addition of⁵¹Cr-labeled (hot) AML target cells inhibited the cytotoxicity of theCD33-CTLs against the “hot” target cells. The ML-2 “cold” targetsexhibit high levels of HLA-A2.1 and costimulatory molecules resulting inthe CD33-CTLs reacting against the cold targets, thereby leaving limitedactivity against the “hot” AML patient cells. The results demonstratethat the cytotoxic activities induced by CD33 peptide-specific CTLs wereCD33+AML cell-specific (FIG. 6).

IFN-γ ELISA

The secretion of cytokines by antigen-specific T cells helps determinetheir effector cell function. IFN-γ secretion by antigen-specific Tcells has been shown to contribute to host defense by initiating apotent local inflammatory response and is an important cytokine againsttumor progression by orienting T lymphocytes into Th1-subtype (Ikeda etal., 2002; Beatty et al., 2001). The CD33 peptide-specific CTLs wereevaluated for secretion of IFN-γ to analyze their Th1 cell subtype withpotential anti-tumor activity. Supernatants from four HLA-A2.1+AIISGDSPV (SEQ ID NO:2)-specific CTL cultures were analyzed for IFN-γproduction after repeated stimulation with the peptide. The amount ofIFN-γ released was significantly higher in the culture supernatants ofCD33-CTLs stimulated with AIISGDSPV (SEQ ID NO:2) pulsed mDCs or T2cells compared to unstimulated control T-cells (Table 5). The resultsdemonstrate that the CD33 peptide-specific CTLs generated with eithermDCs or T2 cells are of a Th-1 subtype and could be potential effectorsagainst CD33+ AML tumor cells.

TABLE 5 Phenotypic analyses of mature dendritic (mDC) and T2antigen-presenting cells. HLA-A2.1 CD80 CD86 CD83 Immature DCs  94  5621  ND¹ DCs matured by LPS 316  71 40 ND DCs matured by TNF-α 475 107 96ND DCs matured by IFN-α + TNF-α 749 137 149  67 T2 cells 577 1214  155 50 ND¹ = not done Phenotypic analysis of culture derived mDC or T2antigen-presenting cells. Immature DCs were generated in vitro fromCD14⁺ monocytes of HLA-A2.1⁺ normal donors by incubation with GM-CSF andIL-4 for 10 days. Immature DCs were treated with LPS, TNF-α, or TNF-α +IFN-α during the last three days of culture to determine the optimalgrowth factor combination required for mature DC generation. Results areexpressed as the mean channel fluorescence (MCF) for each antigentested. Dendritic cells matured with TNF-α + IFN-α displayed the highestlevels of HLA-A2.1, CD80, and CD86 expression compared to LPS or TNF-αcultured DCs. The T2 cell line which can be used as an antigenpresenting cell had the highest level of CD80 expression and similarlevels of HLA-A2.1 and CD86 expression to the TNF-α + IFN-α- cultureddendritic cells.Isotypes of CD33 Peptide-Specific CTLs

Phenotypic analysis of the CD33 peptide-specific CTLs was performed forthe expression of critical antigenic markers including CD8, CD69,CD45RA, CD45RO, and CD28 on the cell surfaces (Table 6). AIISGDSPV (SEQID NO:2) stimulated T cell cultures showed a higher percentage of CD8+cytotoxic T cells or CD69+ activated cells compared to unstimulated Tcells. The percentage of CD8+ cytotoxic T cells was increased byrepeated stimulation of the cultures with CD33 peptide-pulsed mDCs (3cycles=59% vs. 2 cycles=48%). In addition, the percentage of cellsexpressing the CD69 activation antigen increased from 69% after 2stimulations to 82% after three stimulations with CD33 peptide-pulsedmDCs. The percentage of memory cells (CD45RO+) increased dramatically upto 90-95% with a subsequent decrease CD45RA+ naïve cells in the CD33-CTLcultures after three stimulations. The percentage of cells expressingCD28 was similar between CD33 peptide-specific CTLs and unstimulated Tcells implying that the proliferative capacity of the cells wasmaintained in the cultures.

TABLE 6 Phenotype of CD33 peptide-specific CTLs following repeatedstimulation of CTLs with mDC-CD33 peptide. Cell Number of CTLstimulations with mDC-CD33 peptide Antigen Type/Function 0 1 2 3 CD8Cytotoxicity 22 ± 0.71% 48 ± 4.24% 54 ± 7.78% ND¹ CD69 Activation  2 ±1.41%  8 ± 1.41% 39 ± 4.24% 76 ± 9.19% CD45RA Naive T 71 ± 7.07% 57 ±1.41%  8 ± 4.24% ND CD45RO Memory T 52 ± 2.12% 79 ± 0.71% 93 ± 3.54% NDCD28 Proliferation 96 ± 2.12% 81 ± 1.41% 85 ± 3.54% 89 ± 2.12% ND¹ = notdone The phenotype of CD33 peptide specific CTLs was determined by flowcytometry following repeated stimulation of the CTLs with mDCs pulsedwith CD33 peptide. Results are expressed as the mean ± standarddeviation (n = 3).Detection of CD33 Peptide-Recognizing CTLs by Tetramer Staining

The CTL population recognizing AIISGDSPV (SEQ ID NO:2) was characterizedusing AIISGDSPV (SEQ ID NO:2)-HLA-A2.1-tetramers. Peptide-HLA-A2.1tetramers are complexes of four HLA-A2.1 molecules associated with aspecific peptide and a fluorochrome (Altman et al., 1996). The complexesbind to a distinct set of T cell receptors on a subset of CD8+ T cellsrecognizing the specific peptide. CTLs recognizing the AIISGDSPV (SEQ IDNO:2) were identified by staining with AIISGDSPV (SEQ IDNO:2)-HLA-A2.1-tetramers-PE and CD8-FITC antibodies. Flow cytometricanalysis showed that 2.18% of the cells in the AIISGDSPV (SEQ ID NO:2)stimulated cultures was tetramer+CD8+ versus 0.7% or 0% in unstimulatedT cells from HLA-A2.1+or HLA-A2.1-healthy donors, respectively (FIG. 7).

Discussion of Results

In this example, the CD33 epitope was specific to HLA-A2.1, since thisis the most dominant HLA class I molecule; representing approximately50% of North American Caucasians, 34% of African-Americans, and 55% ofAsian-Americans (Baur et al., 1980). The identification of the CD33epitope was performed by the evaluation of the amino acid sequence ofCD33 for peptide motifs, which were likely to bind to HLA-A2.1. Asynthetic peptide homologue of an identified nonamer motif within CD33was examined for the contents to bind to HLA-A2.1 and to elicit thepeptide-specific CTLs by in vitro immunization.

Dendritic cells (DCs) have been used as an APC because of their uniquecapacity to activate naïve T cells and initiate primary antigen-specificT-cell responses (Steinmann A M, 1991; Porgador et al., 1995; Zitvogelet al., 1996). Presentation of antigens by DCs may be especiallyimportant to inducing heightened immune responses to self-antigens sincemany immunization protocols targeting self-antigens have often resultedin the induction of low-affinity CTL responses (Brossart et al., 1996;Houbiers et al., 1993). In this example, mature type of dendritic cellswas used to present exogeneous peptide sufficiently into T cells toevoke the peptide-specific CTLs, thus optimal maturation factors for DCswere examined. Among a few trials, TNF-α and IFN-α induced the bestmaturation, by demonstrating the most up-regulation of HLA-A2.1 andcostimulatory molecules on their cell surfaces by the treatment of thesecytokines. Also, T2 cells, which express significant levels of CD80 andCD86 costimulatory and HLA-A2.1 molecules like DCs, were used as analternative APCs. The results of this study demonstrated that CD33peptide-specific CTLs lysed primary allogeneic AML blasts fromHLA-A2.1+patients or a HLA-A2.1+AML cell line and implies that the CTLsmight be efficiently used as effective cells for immunotherapy in AMLpatients. Low expression of HLA-A2.1 and costimulatory molecules on AMLcells was up-regulated by 48 hours treatment with GM-CSF, IFN-γ andTNF-α. The up-regulation of these molecules was associated withincreased recognition of the AML cells by CD33 peptide-specific CTLs andinduction of higher cytotoxicity by the CTLs. Thus, the prior treatmentof AML patients with these cytokines might improve the recognition ofAML cells by the CTLs and enhance the potential immunotherapy outcome.The present example also demonstrated that DCs and T2 cells efficientlywork as functional APCs to generate CD33 peptide-specific CTLs havingcomparable cytolytic activities. Also, significant amounts of IFN-γ werereleased from the CTLs generated with either DCs or T2 cells-pulsed withCD33 peptide. Thus, these results suggest the usage of T2 cells forfuture clinical application to overcome the limited numbers of DCs andto save the time/efforts to generate DCs.

AML specific cytotoxicity by CD33 peptide-specific CTLs was confirmed inthe cold-target inhibition assay. Complete blocking of the cytolyticactivities was obtained by preincubation with unlabeled AML cells beforethe addition of ⁵¹Cr-labeled bone marrow blasts from AML patients or anAML cell line. The staining of cells with tetramer-PE and anti-CD8antibody-FITC demonstrated that about 2% of stimulated cells are theCD33 peptide-recognizing cytotoxic T lymphocytes. Further phenotypicanalysis showed a higher percentage of CD8 (cytotoxic T), CD45RO(memory), and CD69 (activation) expressing cells following stimulationwith CD33 peptide. Expansion of each cell subtype (CD8, CD45RO, CD69)was observed by repeated stimulation. Also, high levels of CD28expression on CTLs were observed, and it suggests that proliferativecapacity of the stimulated cells will be maintained. Therefore, thisstudy demonstrates the potential efficacy of utilizing CD33-specificpeptide for immunotherapy in AML patients who relapsed after AlloSCT.T-cell based immunotherapy may be accomplished in vivo by the activationand proliferation of anti-leukemia T cells by the administration of CD33peptide alone or dendritic cells pulsed with CD33 peptide or by theinfusion of HLA-compatible anti-leukemia T cell clones generated invitro against the peptide.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” “more than”and the like include the number recited and refer to ranges which can besubsequently broken down into subranges as discussed above. In the samemanner, all ratios disclosed herein also include all subratios fallingwithin the broader ratio.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, thepresent invention encompasses not only the entire group listed as awhole, but each member of the group individually and all possiblesubgroups of the main group. Accordingly, for all purposes, the presentinvention encompasses not only the main group, but also the main groupabsent one or more of the group members. The present invention alsoenvisages the explicit exclusion of one or more of any of the groupmembers in the claimed invention.

All references disclosed herein, including those cited hereafter, arespecifically incorporated herein by reference thereto.

While preferred embodiments have been illustrated and described, itshould be understood that changes and modifications can be made thereinin accordance with ordinary skill in the art without departing from theinvention in its broader aspects as defined in the following claims.

The following are full citations of publications cited in the presentapplication:

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1. An isolated leukemic antigen consisting of a fragment of CD33 antigenselected from the group consisting of: YLALCLCLI (SEQ ID NO: 1),AIISGDSPV (SEQ ID NO: 2), YIISGDSPV (SEQ ID NO: 3), YIISGISPV (SEQ IDNO: 6), YLISGDSPV (SEQ ID NO: 7), ALLALCLCL (SEQ ID NO: 8), TIQLNVTYV(SEQ ID NO: 9), YIGSGDSPV (SEQ ID NO: 10), YIIIGDSPV (SEQ ID NO: 11),YIILGDSPV (SEQ ID NO: 12), YIISGDLPV (SEQ ID NO: 13), YIISGDSWV (SEQ IDNO: 14), YIISGDSPL (SEQ ID NO: 15), ALISGDSPV (SEQ ID NO: 16) andLLALCLCLI (SEQ ID NO: 42).
 2. A pharmaceutical composition comprisingone or more isolated leukemic antigens of claim 1 and a pharmaceuticallyacceptable carrier.
 3. The pharmaceutical composition of claim 2 furthercomprising one or more co-immunostimulatory molecules.